System including biological sample treatment chamber

Abstract

To provide a system for unmasking protein antigens and nucleic acid targets from fixed biological samples.SOLUTION: The present disclosure is directed to specimen processing assemblies including (a) a lower plate 10, (b) an upper plate 30 complementary to the lower plate, and (c) a chamber formed therefrom. In some embodiments, the formed chamber is adapted to perform an unmasking operation, e.g., antigen retrieval and / or target retrieval. In some embodiments, the specimen processing assemblies are configured to keep a specimen-bearing substrate 15 horizontal during all processing steps. The present disclosure is also directed to systems including one or more independently operable specimen processing assemblies.SELECTED DRAWING: Figure 14A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This disclosure claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 891,118, filed August 23, 2019, and the benefit of U.S. Provisional Patent Application No. 62 / 847,388, filed May 14, 2019, the disclosures of which are incorporated herein by reference in their entireties. [Technical Field]

[0002] The present disclosure relates to a system for unmasking protein antigens and nucleic acid targets from fixed biological samples. [Background technology]

[0003] Fixation of tissue and cell samples is used to ensure that the sample's morphology and spatial distribution of biomolecules are preserved, thus helping to enable diagnosis by pathologists. As an adjunct to morphological indicators of disease, the presence of specific proteins and / or nucleic acid sequences can be used to further characterize disease states and, in some cases, to direct disease treatment by oncologists. However, fixation can also interfere with the detection of proteins and nucleic acids in the sample. Therefore, immunohistochemistry (IHC) and in situ hybridization (ISH) procedures often employ an unmasking step (also known as "antigen retrieval" or "target retrieval" for IHC and ISH, respectively) that renders protein antigens or nucleic acid targets accessible to detection reagents such as antibodies or probes.

[0004] Sample fixation is routinely achieved using neutral buffered formalin (NBF). The formaldehyde in NBF preserves tissue and cell morphology by forming crosslinks between reactive groups on proteins and nucleic acids in the sample, and these crosslinks are thought to render certain portions of molecules undetectable. For example, formaldehyde primarily preserves or fixes tissues or cells by crosslinking primary amine groups in proteins with other nearby nitrogen atoms in proteins or DNA via -CH2- bonds. However, the tissue fixation process often masks antigens on specific proteins whose detection is desirable for diagnostic and prognostic purposes. Various methods are used to reverse the effects of formalin fixation and provide access to antigens and targets in fixed biological samples. However, none of these methods are considered completely successful in all cases, and some are more amenable to automation than others. Summary of the Invention

[0005] The present disclosure relates to a specimen processing assembly including (a) a lower plate and (b) an upper plate complementary to the lower plate. The "complementary" upper and lower plates each include features or sets of features that complement each other. For example, the lower plate can have a first set of features (e.g., a substrate stage and a lower engagement surface) and the upper plate can have a second set of features (e.g., a cavity and an upper engagement surface), whereby the first and second sets of features are complementary to each other. In some embodiments, the specimen processing assembly includes complementary lower and upper plates that are independently movable. For example, the lower plate may be coupled to a subassembly that is movable toward the upper plate (e.g., a fixed upper plate or a movable upper plate). As a further example, the upper plate may be coupled to a subassembly that is movable toward the lower plate (e.g., a fixed lower plate or a movable lower plate). In some embodiments, the lower plate has a modular design (a "modular lower plate") that allows it to be used in multiple different specimen processing assemblies.

[0006] In some embodiments, the specimen processing assembly includes a chamber formed from complementary upper and lower plates (or a modular lower plate having features complementary to the upper plate). In some embodiments, the specimen processing assembly is adapted for unmasking, e.g., antigen retrieval and / or target retrieval, of specimens disposed on a substrate and disposed within the chamber. In some embodiments, the specimens disposed on the substrate and / or at least a portion of the substrate itself are maintained at a lower temperature than any other components within the chamber during the unmasking operation. For example, the specimens disposed on the exemplary substrate or a portion of the substrate itself can be maintained as the "coldest" component within the chamber during the unmasking operation. Other aspects of specimen processing assemblies and components of such specimen processing assemblies are further described herein. The present disclosure also relates to systems including one or more independently operable specimen processing assemblies.

[0007] One aspect of the present disclosure is a specimen processing assembly including (a) a lower plate and (b) an upper plate complementary to the lower plate. In some embodiments, the lower plate and the upper plate have complementary polygonal shapes. In some embodiments, the lower plate and the upper plate have complementary wedge shapes. In some embodiments, at least one of the lower plate and the upper plate is movable. In some embodiments, both the lower plate and the upper plate are independently movable.

[0008] In some embodiments, the lower plate includes a first set of features and the upper plate includes a second set of features, the first and second sets of features being complementary to one another. In some embodiments, the first set of features includes a lower engagement surface and one or more substrate stages. In some embodiments, the one or more substrate stages are raised relative to at least a portion of the lower engagement surface. In some embodiments, the one or more substrate stages are recessed relative to at least a portion of the lower engagement surface. In some embodiments, the second set of features includes an upper engagement surface. In some embodiments, the second set of features includes an upper engagement surface and one or more cavities. In some embodiments, the one or more cavities are recessed relative to at least a portion of the upper engagement surface. In some embodiments, each of the lower and upper plates includes additional features, including one or more ports, one or more heating elements, one or more cooling elements, one or more substrate alignment members, etc. In some embodiments, the one or more ports may be intake ports capable of supplying one or more gases and / or vapors. In some embodiments, the one or more ports may be vent ports capable of releasing gases and / or vapors.

[0009] In some embodiments, the lower plate has a modular design. In some embodiments, the modular lower plate includes a body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the body can be mounted on, engaged with, or coupled to the thermal management module. In some embodiments, the body includes a substrate stage that is part of and integral with the body. In some embodiments, the body can be picked up, transported to the thermal management module, and deposited on the thermal management module when the substrate is supported by the body's substrate stage (the body including the substrate stage and the thermal management module together constitute the modular lower plate). In some embodiments, the body is included within a carrier block. In some embodiments, the carrier block and body can be picked up, transported to the thermal management module, and deposited on the thermal management module. In other embodiments, the body includes a lower engagement surface but does not include an integrated substrate stage. Rather, the modular lower plate includes a body with a detachable substrate stage. In this manner, the detachable substrate stage can be used as a carrier for the substrate. For example, a substrate can be placed on a separable substrate stage, and the substrate and separable substrate stage pair can be picked up together, transported to the body, and deposited on the body. In some embodiments, the substrate and separate substrate stage pair remain together through all or at least a portion of processing steps in which a specimen placed on the substrate may be subjected to, for example, cover slip baking or stain baking, through all or a portion of steps used to prepare the sample for microscopic analysis. In some embodiments, a user places a substrate bearing the sample on a separate substrate stage, inputs the substrate / substrate stage pair into the system, and following sample processing, removes the substrate / substrate stage from the system and then removes the substrate bearing the processed sample for analysis.

[0010] In some embodiments, the complementary upper and lower plates include complementary upper and lower engagement surfaces, respectively. In some embodiments, the complementary upper and lower engagement surfaces both include complementary flat surfaces. In some embodiments, the complementary upper and lower engagement surfaces both include complex complementary surfaces, such as complementary surfaces including curved or arcuate shapes. In some embodiments, the complementary upper and lower engagement surfaces are configured such that a sealing engagement can be formed when the upper and lower engagement surfaces at least partially contact one another or a seal disposed therebetween. In some embodiments, the sealing engagement between the complementary upper and lower engagement surfaces facilitates the formation of a sealed chamber.

[0011] In some embodiments, the sealed chamber is heated and / or pressurized for a predetermined amount of time and then cooled. In some embodiments, the sealed chamber is heated and / or pressurized for a predetermined amount of time and then opened without first cooling. In some embodiments, at least a portion of the analyte disposed on the substrate and / or at least a portion of the substrate itself are maintained at a lower temperature than any other components in the sealed chamber during the unmasking operation. For example, the analyte disposed on the substrate or the substrate itself can be maintained as the "coldest" component in the sealed chamber during all steps of the unmasking operation, such as during heating, pressurization, cooling, depressurization, quenching, dispensing of additional fluids, etc. For example, the analyte and / or a portion of the substrate can be maintained at a temperature at least about 2°C lower than any other components in the sealed chamber. As another example, the analyte and / or a portion of the substrate can be maintained at a temperature at least about 5°C lower than any other components in the sealed chamber.

[0012] In some embodiments, any fluids and / or reagents necessary to perform the unmasking operation are first introduced into the specimen disposed on the substrate, a portion of the substrate itself, and / or a reservoir, and then the chamber is sealed. By way of example, before forming the sealed chamber between the upper and lower plates, a total volume of about 250 μL to about 1000 μL of one or more fluids and / or reagents can be dispensed into the specimen disposed on the substrate, a portion of the substrate itself, and / or a reservoir in the lower plate. In some embodiments, no additional fluids and / or reagents are applied after the formation of the sealed chamber.

[0013] In some embodiments, one or more additional fluids and / or reagents are dispensed onto the analyte and / or substrate in the sealed chamber as part of the unmasking operation. For example, about 250 μL to about 1000 μL of one or more additional fluids and / or reagents can be dispensed into the sealed chamber as part of the unmasking operation. In some embodiments, one or more additional fluids and / or reagents are dispensed into the sealed chamber as part of the unmasking operation such that at least about 90% of the chamber's volume is filled with fluids and / or reagents. In some embodiments, one or more additional fluids and / or reagents are dispensed into the sealed chamber as part of the unmasking operation such that at least about 95% of the chamber's volume is filled with fluids and / or reagents. In some embodiments, one or more additional fluids and / or reagents are dispensed into the sealed chamber as part of the unmasking operation such that at least about 99% of the chamber's volume is filled with fluids and / or reagents.

[0014] In some embodiments, either the body of the lower plate or one or more substrate stages is itself configured so that the top surface of the substrate stage is horizontal, for example, parallel to the ground. In embodiments in which the lower plate is movable, the one or more substrate stages and / or the lower body are configured so that the substrate supported by the top surface remains horizontal when the lower plate is moved. In some embodiments, the top surface of the substrate stage remains horizontal throughout the course of the lower plate's movement, regardless of whether the movement of the lower plate remains completely parallel to the ground or whether there is a vertical component to the movement of the lower plate (including embodiments in which there is completely vertical movement). In embodiments in which the bodies of the lower plate and upper plate have complementary wedge shapes, the raised substrate stage itself may have a substantially wedge shape, such that the top surface of the raised substrate stage remains horizontal while the lower plate is moved.

[0015] In some embodiments, the complementary lower and upper plates are independently movable to contact each other at the interface of the complementary lower and upper engagement surfaces. In some embodiments, movement of either the upper or lower plate is influenced by one or more of a motor, a screw, a lever, a spring, a cam mechanism, a piston, or any combination thereof. In some embodiments, after the plates at least partially contact each other, an additional external force is applied to one or both of the upper and / or lower plates. In some embodiments, the application of the additional external force facilitates maintaining the sealing engagement between the upper and lower plates (with or without the use of a seal, as described below). In some embodiments, the external force is applied by one or more force-generating members (e.g., one or more of a motor, a piston, a spring, a screw mechanism, a lever, and / or a cam mechanism).

[0016] In some embodiments, the lower plate and the upper plate simultaneously move toward each other until they contact each other (e.g., their respective upper and lower engagement surfaces contact each other), and then one or more force-generating members are engaged to apply additional force to at least the upper plate. In some embodiments, one or more force-generating members in communication with at least the upper plate can be engaged to press the upper plate against the lower plate, thereby further facilitating sealing engagement between the upper and lower plates or allowing the sealing engagement to be maintained as internal pressure increases within the chamber formed by the upper and lower plates (e.g., during an unmasking operation and / or as internal pressure increases from pre-pressurization).

[0017] In some embodiments, each of the lower plate and the upper plate is independently movable in any of the x, y, and z coordinate directions. In some embodiments, both the lower plate and the upper plate are simultaneously movable toward each other in any of the x, y, and z coordinate directions. In some embodiments, one of the upper plate or the lower plate can be moved to a predetermined position, and simultaneously or subsequently, the other of the upper plate or the lower plate can be moved toward the positioned plate. In some embodiments, the lower plate can be moved to a predetermined position, and then simultaneously or subsequently, the upper plate can be moved toward the positioned lower plate. In some embodiments, the lower plate can be moved to a predetermined position, and then the upper plate can be moved downward toward the lower plate.

[0018] In some embodiments, one of the lower plate or the upper plate is movable, but the other of the lower plate or the upper plate is not movable. In some embodiments, the upper plate is fixed and the lower plate is movable toward the upper plate. In some embodiments, the lower plate is fixed and the upper plate is movable toward the lower plate.

[0019] In some embodiments, the lower plate moves from the loading area to at least one of the preparation area or the unmasking area. In some embodiments, the movement of the lower plate facilitates treatment of substrates supported by the lower plate with one or more fluids and / or reagents. In some embodiments, the lower plate is stationary, and one or more dispensing devices are moved to the stationary lower plate to dispense one or more fluids and / or reagents onto the substrates supported by the lower plate. By way of example, prior to forming a sealed chamber between the lower and upper plates, a total volume of about 250 μL to about 1000 μL of one or more fluids and / or reagents can be dispensed onto the specimen disposed on the substrate, onto a portion of the substrate itself, and / or into a reservoir within the lower plate. In some embodiments, once the substrate is disposed within the sealed chamber, no additional fluids and / or reagents are dispensed onto the substrate.

[0020] In some embodiments, the complementary lower and upper engagement surfaces facilitate sealing engagement without the use of a seal disposed therebetween. In some embodiments, at least one of the lower plate and the upper plate includes at least one seal. In some embodiments, the seal can be disposed within a groove in either the complementary upper and / or lower plate, and the seal, together with the complementary upper and lower engagement surfaces, facilitates sealing engagement between the upper and lower plates. In some embodiments, at least one seal is removable. In some embodiments, the seal can be disposed between the complementary upper and lower engagement surfaces without being disposed within a groove. In some embodiments, the seal is disposed on the surface of the lower engagement surface. In these embodiments, when the upper plate contacts a sealing element disposed on the lower engagement surface, the sealing engagement can be maintained, such as by applying an external force on the lower and / or upper plate using one or more force-generating members. In some embodiments, the at least one removable seal is incorporated into a removable seal attachment, and the removable seal attachment is configured to engage a portion of the periphery of the lower or upper plate. In some embodiments, one of the complementary lower or upper engagement surfaces includes a raised sealing member that protrudes from a plane formed by the one of the complementary lower or upper engagement surfaces, and the other of the complementary lower or upper engagement surface includes a channel that is complementary to the raised sealing member.

[0021] In some embodiments, at least one of the upper plate and the lower plate further includes one or more heating and / or cooling elements. In some embodiments, the cooling element is an active cooling element. In some embodiments, the active cooling element includes a tube at least partially in contact with the body of the lower plate or at least one of the body of the upper plate, and the tube is connected to a circulation device (e.g., a chiller such that a liquid heat transfer medium can be circulated through the tube to cause cooling of at least a portion of the lower plate, the upper plate, and / or a substrate disposed on the substrate stage). In some embodiments, the cooling element is a passive cooling element. In some embodiments, the passive cooling element is a heat sink. In some embodiments, only the upper plate includes a heating element. In some embodiments, at least one of the upper plate or the lower plate includes a heating element, and the lower plate includes a passive or active cooling element. In some embodiments, only the lower plate includes a heating element.

[0022] In embodiments in which both the upper and lower plates include heating and / or cooling elements, the heating and / or cooling elements may be independently operable, as described herein. In some embodiments, any of the heating and / or cooling elements in the upper plate can be operated in conjunction with the heating and / or cooling elements in the lower plate or those embedded in one or more thermal management modules. As a result, by independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate stage, any portion of the substrate supported by the substrate stage, and / or the chamber can be controlled. In some embodiments, independent control of the various heating and / or cooling elements allows a temperature gradient, for example, a temperature gradient between the substrate supported by the substrate stage and the interior walls of the formed chamber, to be formed and maintained.

[0023] In some embodiments, at least one of the upper plate and the lower plate further includes a sensor, such as a temperature sensor. In some embodiments, the upper plate includes a temperature sensor in contact with the substrate and / or a sample disposed on the substrate. In some embodiments, the lower plate includes a temperature sensor in contact with the substrate. In some embodiments, data from the temperature sensor is monitored so that one or more heating and / or cooling elements disposed in the lower and / or upper plates can be controlled. For example, the sensor can provide feedback so that one or more heating and / or cooling elements disposed in the lower and / or upper plates can be controlled to maintain a portion of the analyte or substrate as the coldest component in the sealed chamber.

[0024] Another aspect of the present disclosure is a specimen processing assembly including: (i) a lower plate coupled to a subassembly, the lower plate including a lower engagement surface and one or more substrate stages, the one or more substrate stages including an upper surface adapted to hold a substrate horizontally; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface. In some embodiments, the upper plate further includes one or more cavities. In some embodiments, the one or more cavities each receive at least a portion of the one or more substrate stages. In some embodiments, one of the lower plate and the one or more substrate stages is configured such that any substrates supported by the one or more substrate stages remain parallel to the ground throughout movement of the lower plate along the subassembly, and the upper surface of the one or more substrate stages remains horizontal as the lower plate traverses the subassembly (e.g., regardless of whether the movement is completely horizontal along the subassembly or whether the movement includes a vertical component to the movement, or even purely vertical movement). In some embodiments, the substrate is a microscope slide.

[0025] In some embodiments, the lower plate is coupled to a subassembly that functions to move the lower plate into contact with the upper plate. In some embodiments, the subassembly is moved with sufficient force so that movement of the subassembly itself is sufficient to form a seal between the lower plate and the upper plate coupled to the subassembly. For example, the subassembly can include a means for translating the lower plate along a rail (e.g., a horizontally disposed rail or, as described below, a rail having one end offset from the horizontal). In some embodiments, the subassembly includes a motor adapted to move the lower plate along the rail, e.g., from a loading position to an unmasking position.

[0026] In some embodiments, the subassembly is positioned horizontally. In this configuration, the lower plate moves along the horizontally positioned subassembly. In some embodiments, the subassembly is positioned such that a first end of the subassembly is vertically raised relative to a second end of the subassembly, e.g., the subassembly is offset from horizontal. In this particular embodiment, when the lower plate is moved along the subassembly, e.g., when the lower plate is moved along either the x-direction and / or y-direction of the horizontally offset subassembly, the lower plate moves with a motion that includes a vertical component, and given the horizontal offset of the rail, there is a concomitant movement in the z-direction. In some embodiments, the rail is offset from horizontal at an angle ranging from about 5 degrees to about 70 degrees. In other embodiments, the horizontal offset angle is ranging from about 5 degrees to about 60 degrees. In yet other embodiments, the horizontal offset angle is ranging from about 10 degrees to about 50 degrees. In further embodiments, the horizontal offset angle is ranging from about 20 degrees to about 50 degrees. In yet other embodiments, the horizontal offset angle is ranging from about 20 degrees to about 45 degrees. In some embodiments, the subassembly further includes a motor adapted to move the lower plate along the subassembly.

[0027] In some embodiments, the upper plate is coupled to the support member, and the lower plate is moved (along either the horizontal subassembly or the horizontally offset subassembly) to a position where the lower engagement surface contacts a complementary upper engagement surface on the upper plate. In some embodiments, the upper plate is coupled to one or more springs, and the lower plate is moved to a position where the lower engagement surface contacts a complementary upper engagement surface on the upper plate, and the one or more springs apply a downward force to the upper plate as the lower plate contacts the upper plate.

[0028] In some embodiments, the lower plate is moved to a predetermined position below the upper plate, and the upper plate is moved toward the lower plate positioned at least along the z-axis. In other embodiments, the lower plate is moved near the upper plate, and the upper plate is simultaneously or subsequently moved toward the lower plate (in any of the x, y, and z directions) until the upper engagement surface of the upper plate contacts the lower engagement surface of the lower plate. In still other embodiments, the lower plate and the upper plate are both moved toward each other simultaneously (wherein the respective movements of the lower plate and the upper plate can independently be in any of the x, y, and / or z directions). In some embodiments, the lower plate and the upper plate are both moved toward each other simultaneously until they both contact each other (e.g., their respective upper and lower engagement surfaces contact each other), and then a force-generating member in communication with the upper plate is engaged. In some embodiments, engagement of the force-generating member causes a force to be exerted on at least the upper plate. In some embodiments, the force exerted on the upper plate facilitates sealing engagement between the upper plate and the lower plate. In some embodiments, a predetermined amount of force is applied to the upper plate by the force-generating member. In some embodiments, the predetermined amount of force applied to the upper plate by the force-generating member is greater than the force generated by internal pressurization of the chamber formed by the upper and lower plates, but less than a predetermined threshold force that could generate unsafe pressure within the chamber. In some embodiments, the predetermined force applied by the force-generating member is limited such that if the pressure within the chamber exceeds the predetermined pressure, the force applied by the force-generating member is overcome and the force-generating member slips or releases pressure above the predetermined pressure that could be generated within the chamber.

[0029] In some embodiments, the specimen processing assembly includes a chamber. In some embodiments, the chamber is formed from complementary lower and upper plates. In some embodiments, complementary lower and upper engagement surfaces of the lower and upper plates, respectively, facilitate sealing engagement between the upper and lower plates such that a chamber is formed between the upper and lower plates. In some embodiments, the complementary lower and upper engagement surfaces provide sealing engagement without the use of a seal disposed therebetween. In some embodiments, a seal can be disposed within a groove in either the upper and / or lower plate, and the seal, together with the upper and lower engagement surfaces, facilitates sealing engagement between the upper and lower plates. In some embodiments, the seal may be disposed between the upper and lower engagement surfaces without being disposed within a groove, e.g., the seal is on the surface of the lower engagement surface. In some embodiments, one of the lower or upper engagement surfaces includes a raised seal member protruding from a plane formed by the one of the lower or upper engagement surfaces, and the other of the lower or upper engagement surface includes a channel complementary to the raised seal member.

[0030] In some embodiments, one or both of the lower plate and the upper plate include one or more heating and / or cooling elements. In some embodiments, the one or more heating and / or cooling elements in the lower and upper plates are operated together, for example, to establish and maintain a predetermined temperature gradient within the chamber. For example, the one or more heating and / or cooling elements in the lower and upper plates are operated together to establish and maintain a temperature gradient between the substrate stages and other components within the chamber, such as between walls defining the interior of the chamber. In some embodiments, the one or more heating and / or cooling elements are independently controlled so that a analyte disposed on the substrate and / or a portion of the substrate itself is maintained at a lower temperature than any other components within the chamber (e.g., chamber walls, ports, valves, sensors, probes, etc.). For example, a portion of the analyte and / or substrate can be maintained at a temperature at least about 2°C lower than any other components within the sealed chamber. As another example, a portion of the analyte and / or substrate can be maintained at a temperature at least about 5°C lower than any other components within the sealed chamber. In some embodiments, the upper plate further includes one or more ports. In some embodiments, the one or more ports allow for the introduction of one or more gases and / or vapors into the chamber formed from the lower plate and the upper plate. For example, steam can be introduced to heat the analyte in the chamber and simultaneously pressurize the chamber. In some embodiments, the upper plate further includes one or more valves to facilitate the release of gases and / or vapors from within the chamber formed from the lower plate and the upper plate. For example, the one or more valves can be opened for a predetermined amount of time to allow pressure to be released from the sealed chamber. Alternatively, the sealed chamber can be opened immediately after the unmasking operation is completed, for example, the sealed chamber can be opened without first cooling or depressurizing.

[0031] In some embodiments, the unmasking operation performed within the chamber formed from the complementary bottom and top plates is performed using fluids and / or reagents that are supplied to the substrate prior to formation of the chamber, hi some embodiments, no additional fluids and / or reagents are dispensed onto the substrate after the substrate is placed within the chamber and the chamber is sealed.

[0032] In some embodiments, the chamber can include one or more ports through which one or more additional fluids and / or reagents can be added directly to the sealed chamber, such as as part of an unmasking operation. In some embodiments, the one or more additional fluids and / or reagents are dispensed onto the specimen and / or substrate in the sealed chamber as part of the unmasking operation. For example, about 250 μL to about 1000 μL of one or more additional fluids and / or reagents can be dispensed into the sealed chamber as part of the unmasking operation. In some embodiments, the one or more additional fluids and / or reagents are dispensed into the sealed chamber as part of the unmasking operation such that at least about 90% of the volume of the chamber is filled with fluids and / or reagents. In some embodiments, the lower plate can include one or more ports, such as a vacuum port, for removing excess liquid from within the chamber. In some embodiments, the vacuum port of the lower plate can be controlled after the chamber has been substantially filled with the additional fluids and / or reagents.

[0033] Another aspect of the present disclosure is a system including one or more independently operable specimen processing assemblies, one or more dispensing devices, one or more optional liquid removal devices, one or more optional mixing devices, and a control system communicatively coupled to the one or more specimen processing assemblies and at least one or more dispensing devices. In some embodiments, each of the one or more independently operable specimen processing assemblies includes one or more lower plates and one or more upper plates, each of the one or more upper plates being complementary to each of the one or more lower plates.

[0034] In some embodiments, the lower plate has a modular design. In some embodiments, the modular lower plate includes a body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the body can be mounted on, engaged with, or coupled to the thermal management module. In some embodiments, the body includes a substrate stage that is part of and integral with the body. In other embodiments, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body with a separable substrate stage. In this manner, the separable substrate stage can be used as a carrier for the substrate. For example, a substrate can be placed on the separable substrate stage, and the substrate and separable substrate stage pair can be picked up together, transported to the body, and deposited on the body. In some embodiments, the substrate and separate substrate stage pair remain together throughout all or at least a portion of the processing steps used to prepare the sample for microscopy, such as, for example, cover slip baking or stain baking. In some embodiments, a user places a substrate holding a sample on a separate substrate stage, inputs the substrate / substrate stage pair into the system, and following sample processing, removes the substrate / substrate stage from the system and then removes the substrate with the processed sample for analysis.

[0035] In some embodiments, the system includes at least two independently operable specimen processing assemblies, one of which includes a sealed chamber (e.g., a chamber formed from one upper plate and one complementary lower plate or complementary modular lower plates).

[0036] In some embodiments, the lower plate and the upper plate include one or more independently operable heating and / or cooling elements. For example, independently operable heating and / or cooling elements may be present in the lower plate, the upper plate, or both the lower plate and the upper plate. In some embodiments, the chamber is in communication with at least two heating and / or cooling elements. In some embodiments, the heating and / or cooling elements in the upper plate can be operated in conjunction with the heating and / or cooling elements in the lower plate. By independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate stage, any portion of the substrate supported by the substrate stage, and / or the chamber can be controlled.

[0037] In some embodiments, at least two heating and / or cooling elements may establish a thermal gradient between different portions of the lower and / or upper plates. In some embodiments, the established thermal gradient may enable the substrate and / or analytes disposed on a portion of the substrate to be maintained at a lower temperature than the temperature of any other components in the chamber during the unmasking operation. For example, various heating and / or cooling elements present on the lower and / or upper plates may be independently operated such that at least a portion of the substrate, or an analyte disposed on the substrate, remains the coldest component in the chamber during the unmasking operation. For example, the substrate or an analyte disposed on the substrate may be maintained at a lower temperature than the upper plate, the lower plate, ports, valves, and / or any other structures in the chamber formed from the upper and lower plates during the unmasking operation.

[0038] In some embodiments, the duration of the unmasking operation is standardized for all types of specimens and all types of analyses performed on a given type of specimen, thereby increasing the efficiency of the system and all components therein. For example, having a standardized duration for all specimens and analysis types allows for easier scheduling of previous specimen processing steps (e.g., deparaffinization) and subsequent specimen processing steps (e.g., staining steps), since all can be in "lock-step" with the standardized unmasking operation duration. Standardization of the unmasking operation duration is made possible by the discovery that it is only necessary to change the temperature to vary the degree of unmasking for a given type of sample. By selecting a specific temperature at which the unmasking operation is performed for a particular sample type, not only can the duration of the unmasking operation be standardized, but the degree of unmasking for a particular sample type can be optimized for a particular assay. In some embodiments, the disclosed system can be optimized to facilitate standardization of the duration of the unmasking operation. Thus, in some embodiments, the unmasking operation in each independently operable chamber is performed for the same duration, regardless of whether biomarkers are unmasked in the individual chamber.

[0039] Another aspect of the present disclosure is a system including one or more independently operable analyte processing assemblies adapted to independently process and / or move analyte holding substrates from one processing region to another while maintaining the analyte holding substrates in a horizontal position during all processing steps (e.g., during treatment with one or more fluids and / or reagents and during unmasking operations). In some embodiments, the horizontal processing (and / or horizontal movement) allows one or more fluids and / or reagents dispensed on the analyte holding substrate to be maintained and / or manipulated on the surface of the substrate. In some embodiments, the one or more analyte processing assemblies include a chamber configured to process analyte holding substrates at elevated temperatures and / or pressures (compared to the temperature and / or pressure outside the chamber) while maintaining the substrate in a horizontal position and minimizing evaporative loss. In some embodiments, the chamber is formed from a lower plate having a lower engagement surface and an upper plate having an upper engagement surface, the upper engagement surface being complementary to the lower engagement surface.

[0040] In some embodiments, the chamber is formed from a modular lower plate. In some embodiments, the modular lower plate includes a body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the body can be mounted on, engaged with, or coupled to the thermal management module. In some embodiments, the body includes a substrate stage that is part of and integral with the body. In other embodiments, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body with a separable substrate stage. In this manner, the separable substrate stage can be used as a carrier for the substrate. For example, a substrate can be placed on the separable substrate stage, and the substrate and separable substrate stage pair can be picked up together, transported to the body, and deposited on the body. In some embodiments, the substrate and separate substrate stage pair remain together throughout all or at least a portion of a processing step, e.g., throughout all or a portion of a step used to prepare a sample for microscopy, such as cover slip baking or stain baking. In some embodiments, a user places a substrate holding a sample on a separate substrate stage, inputs the substrate / substrate stage pair into the system, and following sample processing, removes the substrate / substrate stage from the system and then removes the substrate with the processed sample for analysis.

[0041] In some embodiments, the lower plate and the upper plate are moved independently of one another such that sealing engagement occurs between the complementary lower and upper engagement surfaces. In some embodiments, the formed chamber is configured to perform one or more steps of an unmasking operation, such as heating, pressurizing, cooling, depressurizing, quenching, and / or adding or removing fluids and / or reagents.

[0042] In some embodiments, one or more fluids and / or reagents are dispensed onto the substrate, a portion of the substrate itself, and / or a specimen disposed in a reservoir in the lower plate prior to forming the sealed chamber. For example, a total volume of about 250 μL to about 1000 μL of one or more fluids and / or reagents is dispensed onto the specimen disposed on the substrate, a portion of the substrate itself, and / or a reservoir in the lower plate prior to forming the sealed chamber. In some embodiments, the unmasking operation is performed using the fluids and / or reagents provided to the substrate prior to forming the chamber. For example, no additional fluids and / or reagents are dispensed onto the substrate while the substrate is disposed in the chamber and during the unmasking operation.

[0043] In some embodiments, the chamber is in communication with one or more independently operable heating and / or cooling elements that allow for selective heating and / or cooling of the substrate, the specimen, and / or one or more fluids and / or reagents disposed within the chamber to a predetermined temperature. In some embodiments, heating of a fluid (such as a fluid disposed on the substrate or in a separate reservoir within the chamber) allows the chamber to be heated and / or pressurized. For example, heating of the fluid can allow an unmasking operation to be performed on the specimen disposed within the sealed chamber and on the substrate. In some embodiments, heating and / or pressurization of the chamber is monitored by one or more temperature and / or pressure sensors disposed within the chamber or in communication with at least one of the specimen or substrate. In some embodiments, heating is achieved by conductive heating elements disposed on or within the substrate stage, conductive heating elements within the body of one or both of the upper and lower plates, other types of heating devices located adjacent to the fluids and / or reagents to be heated, using microwaves passed through the reaction chamber to heat the reagents, and / or magnetic induction. In some embodiments, at least a portion of the analyte disposed on the substrate and / or at least a portion of the substrate itself is maintained at a lower temperature than any other component in the chamber during the unmasking operation. For example, the analyte disposed on the substrate or the substrate itself can be maintained as the "coldest" component in the chamber during the unmasking operation.

[0044] In some embodiments, the sealed chamber is heated to a predetermined temperature and / or pressurized to a predetermined pressure for a predetermined time. For example, the sealed chamber can be heated to a temperature ranging from about 115° C. to about 155° C. and a pressure ranging from about 150 kPa to about 1050 kPa for a time ranging from about 2 minutes to about 10 minutes. In some embodiments, a portion of the specimen and / or substrate is maintained as the "coldest" component within the chamber throughout the heating and / or pressurization process.

[0045] In some embodiments, the unmasking operation includes a temperature increase phase, a temperature maintenance phase, and a temperature decrease phase. In some embodiments, a substrate disposed on the substrate or a portion of the substrate itself is maintained as the "coldest" component in the chamber during the temperature increase phase, the temperature maintenance phase, and the temperature decrease phase. In some embodiments, the unmasking operation does not include a temperature decrease phase. For example, the sealed chamber may be opened while heated and / or pressurized. In some embodiments, prior to opening the heated and / or pressurized chamber, a portion of the pressure is released via one or more valves communicating with the chamber.

[0046] In some embodiments, additional fluids and / or reagents are added to the specimen and / or substrate during one or more of the temperature increase, temperature maintenance, and temperature decrease phases. For example, about 250 μL to about 1000 μL of one or more additional fluids and / or reagents are dispensed onto a portion of the specimen or substrate itself after the chamber is formed. In some embodiments, the one or more additional fluids and / or reagents dispensed into the sealed chamber have a temperature lower than the temperature within the heated and / or pressurized chamber. For example, the one or more additional fluids and / or reagents can have a temperature at least 25° C. lower than the temperature of the specimen or the temperature within the sealed chamber. In some embodiments, the entire sealed chamber is substantially filled with one or more liquids. For example, if the total chamber volume is about 2 mL (not considering the volume of the substrate containing the chamber), about 2 mL of additional fluids and / or reagents can be added while the chamber is sealed. In some embodiments, about 250 μL to about 1000 μL of one or more additional fluids and / or reagents are dispensed into a reservoir within the chamber, such as a heated fluid reservoir. In an alternative embodiment, the fluid reservoir is maintained as the coldest component along with the chamber.

[0047] In some embodiments, the chamber is in communication with one or more ports that facilitate delivery of one or more gases and / or vapors into the chamber to pressurize the chamber and / or heat the substrate, analyte, and / or one or more fluids and / or reagents disposed within the chamber. In some embodiments, the lower plate can include one or more ports, e.g., vacuum ports, for removing excess liquid from within the chamber. In some embodiments, the vacuum ports in the lower plate can be activated after the chamber has been substantially filled with additional fluids and / or reagents.

[0048] Another aspect of the present disclosure is a system including: (a) one or more independently operable specimen processing assemblies, the one or more independently operable specimen processing assemblies including: (i) a lower plate including a lower engagement surface and one or more substrate stages raised relative to the lower engagement surface, the one or more raised substrate stages including an upper surface adapted to hold a substrate horizontally; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface, the upper plate further including one or more cavities recessed relative to the upper engagement surface, the one or more recessed cavities adapted to receive at least a portion of the raised substrate stages; (b) one or more dispensing devices; and (c) a control system communicatively coupled to at least the one or more independently operable specimen processing assemblies and / or the one or more dispensing devices.

[0049] In some embodiments, at least one of the lower plate and the upper plate includes one or more independently operable heating and / or cooling elements. For example, the lower plate can include one, two, or three independently operable heating and / or cooling elements. The upper plate can include one heating and / or cooling element. In some embodiments, at least one heating and / or cooling element is embedded within the body of the lower plate. At least one heating and / or cooling element is embedded within the body of the upper plate. In some embodiments, a control system is in communication with the one or more independently operable heating and / or cooling elements. In some embodiments, any of the heating and / or cooling elements in the upper plate can be operated in conjunction with the heating and / or cooling elements in the lower plate. By independently controlling the various heating and / or cooling elements present on the upper and lower plates, the temperature of the substrate stage, any portion of the substrate supported by the substrate stage, and / or the chamber can be controlled. For example, various heating and / or cooling elements present on the lower and / or upper plates can be independently operated so that at least a portion of the substrate, or a specimen disposed thereon, remains the coldest structure within the chamber. For example, the substrate or a specimen disposed thereon can be maintained at a lower temperature than the upper and lower plates, ports, valves, and / or any other structures within the chamber formed from the upper and lower plates. In some embodiments, the heating and / or cooling elements are independently operated to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0050] In some embodiments, one or more independently operable heating and / or cooling elements are in thermal communication with one or more substrate stages, hi some embodiments, the independently operable heating and / or cooling elements are configured such that an analyte disposed on the substrate and disposed on a surface of the substrate stage has a lower temperature than any other component in thermal communication with the one or more independently operable heating and / or cooling elements.

[0051] In some embodiments, each dispenser of the one or more dispensers includes one or more dispense nozzles, one or more pipettes, and / or one or more on-demand dispensers. For example, the dispensers can be commanded by the control system to dispense a total volume of one or more fluids and / or reagents ranging from about 250 μL to about 1000 μL. In some embodiments, the system further includes one or more mixing devices and / or one or more liquid removal devices. In some embodiments, the one or more dispensers are coupled to a dispense subassembly. In some embodiments, the system includes multiple specimen processing assemblies. In some embodiments, at least one of the multiple specimen processing assemblies includes a chamber formed from an upper plate and a lower plate. In some embodiments, the substrate is positioned horizontally within the chamber.

[0052] Another aspect of the present disclosure is a system including: (a) a plurality of independently operable specimen processing assemblies, each of the independently operable specimen processing assemblies including: (i) a lower plate movably coupled to a lower rail, the lower plate including a lower engagement surface and one or more substrate stages raised relative to the lower engagement surface, the one or more raised substrate stages including an upper surface adapted to hold a substrate horizontally as the lower plate traverses the lower rail; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface, the upper plate further including one or more cavities recessed relative to the upper engagement surface, the one or more recessed cavities adapted to receive at least a portion of the one or more raised substrate stages, the lower plate configured to traverse the lower rail while the planar upper surface of the substrate stages remains horizontal; (b) one or more dispensers; and (c) a control system in communication with the plurality of independently operable specimen processing assemblies and / or the one or more dispensers.

[0053] In some embodiments, the control system is adapted to maintain an environment within a chamber formed from complementary lower and upper plates, such as by controlling one or more heating elements, one or more cooling elements, and / or one or more gas and / or vapor generation and delivery units in communication with the chamber. In some embodiments, the one or more heating and / or cooling elements are independently controlled so that analytes disposed on the substrate and / or portions of the substrate itself are maintained at a lower temperature than any other components (e.g., ports, valves, sensors, probes, etc.) within the formed chamber. In some embodiments, the control system monitors the temperature and / or pressure within the chamber (e.g., using one or more temperature and / or pressure sensors within the chamber and / or in contact with the substrate) and increases and / or decreases the temperature and / or pressure to maintain a predetermined temperature and / or pressure. For example, the temperature within the chamber can be monitored by a temperature sensor communicatively coupled to the control system. When the temperature within the chamber meets a predetermined threshold chamber temperature, the control system can command one or more heating elements to maintain a steady state or to turn off.

[0054] In some embodiments, the control system directs one or more force-generating members to apply a predetermined external force to at least one of the upper plate or the lower plate to maintain the respective upper and lower engagement surfaces of the upper and lower plates in sealing engagement, particularly when pressure in any formed chamber increases. In some embodiments, the control system directs any force-generating members to apply a predetermined amount of force that is less than the force generated at a predetermined threshold pressure. In some embodiments, the control system can direct one or more ports and / or valves to release pressure above the predetermined threshold pressure.

[0055] In some embodiments, the one or more substrate stages are configured such that any substrate supported by the one or more substrate stages is held in a horizontal position and remains in a horizontal position during movement of the lower plate within the system, regardless of whether the movement is entirely horizontal or whether the movement includes a vertical component (or even entirely vertical movement).

[0056] In some embodiments, at least one of the analyte processing assemblies includes a sealed chamber. In some embodiments, the system further includes a substrate at least partially disposed within the sealed chamber, the substrate supported by one or more substrate stages and oriented in a horizontal position. In some embodiments, the analyte disposed on the substrate is treated with one or more fluids and / or reagents outside the chamber while in the horizontal position, and an unmasking operation is performed within the chamber while the substrate is disposed in the horizontal position. In some embodiments, no additional fluids and / or reagents are dispensed onto the substrate while the substrate is disposed within the chamber. In some embodiments, the unmasking operation is performed using only fluids and / or reagents dispensed onto the substrate prior to formation of the chamber. In some embodiments, the analyte disposed on the substrate or the substrate itself is maintained as the “coldest” component within the chamber during all stages of the unmasking operation. For example, the substrate and / or a portion of the substrate is maintained as the “coldest” component within the chamber during the temperature increase, temperature maintenance, and temperature decrease stages of the unmasking operation.

[0057] In some embodiments, the lower rail of each of the plurality of independently operable specimen processing assemblies is disposed horizontally, e.g., each lower rail is disposed parallel to the ground. In some embodiments, the lower rail of each of the plurality of independently operable specimen processing assemblies includes a first end that is raised perpendicularly relative to a second end, such that each lower rail deviates from horizontal, e.g., the rail is offset from horizontal.

[0058] In some embodiments, the system further includes a motor adapted to move the lower plate from a first position along the lower rail to a second position along the lower rail. In some embodiments, the second position along the lower rail is a predetermined position below the upper plate, e.g., below a pre-positioned upper plate. In some embodiments, the second position along the first lower rail is within a preparation area, and the upper plate is moved (along any of the x, y, and z directions) to the pre-positioned lower plate.

[0059] In some embodiments, the distribution device is coupled to a distribution rail, which is oriented vertically relative to the lower rails of each of the multiple independently operable specimen processing assemblies (regardless of whether the lower rails are horizontally disposed or offset from horizontal). In some embodiments, the upper plate is coupled to a force-generating member. In some embodiments, the force-generating member is selected from the group consisting of a lever, a screw, a motor, a spring, a pneumatic piston, a hydraulic piston, a cam mechanism, and any combination thereof. In some embodiments, the upper plate is fixed to the subassembly.

[0060] In some embodiments, the disclosed system includes at least two independently operable specimen processing assemblies, hi some embodiments, one of the at least two independently operable specimen processing assemblies includes a chamber.

[0061] Another aspect of the present disclosure is a specimen processing assembly including: (i) a lower plate including: (a) a substrate stage having an upper surface adapted to horizontally support a substrate; (b) a lower engagement surface at least partially circumscribing the substrate stage; and (c) a first lower temperature adjustment element in thermal communication with the substrate stage; and (ii) an upper plate including: (a) an upper engagement surface complementary to the lower engagement surface; and (b) a cavity. In some embodiments, the first lower temperature adjustment element is disposed below the substrate stage. In some embodiments, the lower plate further includes a second lower temperature adjustment element and a third lower temperature adjustment element. In some embodiments, each of the temperature adjustment elements is independently operable. In some embodiments, the second lower temperature adjustment element and the third lower temperature adjustment element are each disposed adjacent to the first lower temperature adjustment element. In some embodiments, the second lower temperature adjustment element and the third lower temperature adjustment element are each disposed below a portion of the lower engagement surface. In some embodiments, the first lower engaging element, the second lower engaging element, and the third lower engaging element are arranged parallel to one another.

[0062] In some embodiments, a temperature gradient is maintained between the first and second lower temperature adjustment elements and between the first and third temperature adjustment elements. In some embodiments, the temperature gradient maintained between the first and second lower temperature adjustment elements is in the range of 2°C to about 10°C. The temperature gradient maintained between the first and second lower temperature adjustment elements is in the range of 2°C to about 10°C. In some embodiments, the temperature gradient maintained between the first and second lower temperature adjustment elements is in the range of 2°C to about 5°C. The temperature gradient maintained between the first and second lower temperature adjustment elements is in the range of 2°C to about 5°C. In some embodiments, the first, second, and third lower temperature adjustment elements are positioned to maintain a temperature gradient between at least a portion of the lower engagement surface and the substrate stage.

[0063] In some embodiments, the first lower temperature adjustment element has a first heat output, the second lower temperature adjustment element has a second heat output, and the third lower temperature adjustment element has a third heat output, the first heat output being less than either the second heat output or the third heat output. In some embodiments, the upper plate further includes at least one upper heat adjustment element. In some embodiments, the heat output of the first lower temperature adjustment element is maintained at a temperature lower than the heat output of the at least one upper temperature adjustment element.

[0064] In some embodiments, the analyte processing assembly further includes a substrate disposed on a surface of the substrate stage. In some embodiments, the substrate is maintained at a temperature lower than the temperatures of the lower and upper engagement surfaces. In some embodiments, the analyte disposed on the substrate or a portion of the substrate is maintained at a temperature lower than any other component in the chamber, e.g., the analyte disposed on the substrate or a portion of the substrate is the "coldest" component in the chamber during the unmasking operation.

[0065] In some embodiments, the first lower temperature adjustment element includes at least one fluid channel. In some embodiments, the second lower temperature adjustment element and the third lower temperature adjustment element each include a heating cartridge. In some embodiments, at least one of the lower plate and the upper plate includes at least one seal. In some embodiments, the at least one seal is removable. In some embodiments, at least one of the upper plate or the lower plate is coupled to at least one of a motor, a piston, a spring, a screw mechanism, a lever, or a cam mechanism.

[0066] In some embodiments, the specimen processing assembly further includes a subassembly having a first end and a second end, hi some embodiments, the lower plate is movable along the length of the subassembly between the first end and the second end.

[0067] In some embodiments, the subassembly is positioned horizontally. In some embodiments, the lower plate and the upper plate are both independently moved into position such that the lower engagement surface of the lower plate at least partially contacts the complementary upper engagement surface of the upper plate. In some embodiments, the lower plate is moved into position and then the upper plate is moved toward the lower plate. In some embodiments, the movement toward the lower plate is performed using one of a motor, a piston, or a cam mechanism.

[0068] In some embodiments, the subassembly is offset from horizontal. In some embodiments, the upper plate is held stationary and the lower plate is moved toward the upper plate until the lower engagement surface of the lower plate at least partially contacts the complementary upper engagement surface of the upper plate. In some embodiments, the upper and lower plates have complementary wedge shapes. In some embodiments, the lower and upper plates are both independently moved into position such that the lower engagement surface of the lower plate at least partially contacts the complementary upper engagement surface of the upper plate. In some embodiments, the lower and upper plates are both independently movable.

[0069] Another aspect of the present disclosure is a method for unmasking an analyte disposed on a substrate, the method comprising: (a) dispensing a predetermined amount of one or more fluids and / or reagents onto at least a portion of the analyte; (b) sealing the analyte in a chamber, the chamber being formed by contacting (i) a lower engagement surface of a lower plate with (ii) an upper engagement surface of an upper plate, the upper engagement surface being complementary to the lower engagement surface, and the lower plate further comprising a substrate stage for supporting the substrate; and (c) performing an unmasking operation on the analyte in the sealed chamber. In some embodiments, the lower plate includes one or more independently operable heating and / or cooling elements. In some embodiments, the upper plate includes one or more independently operable heating and / or cooling elements. In some embodiments, both the lower and upper plates include operable heating and / or cooling elements. In some embodiments, either the heating and / or cooling elements in the upper plate can be operated together with the heating and / or cooling elements in the lower plate. By independently controlling the various heating and / or cooling elements present on the upper and lower plates, the temperature of the substrate stage, any portion of the substrate supported by the substrate stage, and / or the chamber can be controlled.

[0070] In some embodiments, the chamber is formed from a lower plate having a modular design. In some embodiments, the modular lower plate includes a body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the body can be mounted on, engaged with, or coupled to the thermal management module. In some embodiments, the body includes a substrate stage that is part of and integral with the body. In other embodiments, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body with a separable substrate stage. In this manner, the separable substrate stage can be used as a carrier for the substrate. For example, a substrate can be placed on the separable substrate stage, and the substrate and separable substrate stage pair can be picked up together, transported to the body, and deposited on the body. In some embodiments, the substrate and separate substrate stage pair remain together throughout all or at least a portion of a processing step, e.g., throughout all or a portion of a step used to prepare a sample for microscopy, such as cover slip baking or stain baking. In some embodiments, a user places a substrate holding a sample on a separate substrate stage, inputs the substrate / substrate stage pair into the system, and following sample processing, removes the substrate / substrate stage from the system and then removes the substrate with the processed sample for analysis.

[0071] In some embodiments, the one or more fluids and / or reagents are selected from water and a buffer having a pH ranging from about 5 to about 10. In some embodiments, the one or more fluids and / or reagents comprise a mixture of deionized water, tris(hydroxymethyl)methylamine, and a chelating agent. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto at least a portion of the specimen ranges from about 200 μL to about 1000 μL. In some embodiments, the predetermined amount of the one or more fluids and / or reagents dispensed onto at least a portion of the specimen ranges from about 250 μL to about 500 μL.

[0072] In some embodiments, the unmasking operation includes heating the specimen disposed on the substrate to a first predetermined temperature for a predetermined period of time. In some embodiments, the first predetermined temperature is in a range from about 125°C to about 155°C. In some embodiments, the first predetermined temperature is in a range from about 135°C to about 150°C. In some embodiments, the first predetermined temperature is about 140°C. In some embodiments, the predetermined duration is in a range from about 1 minute to about 10 minutes. In some embodiments, the predetermined duration is in a range from about 1 minute to about 7 minutes. In some embodiments, the predetermined duration is in a range from about 1 minute to about 5 minutes.

[0073] In some embodiments, the temperature of the substrate is lower than the temperature of any other components in the chamber during the unmasking operation. In some embodiments, the temperature of the substrate is at least 10° C. lower than the temperature of the other components in the chamber. In some embodiments, the temperature of the substrate is at least 5° C. lower than the temperature of the other components in the chamber.

[0074] In some embodiments, less than about 5% of the predetermined amount of one or more fluids and / or reagents dispensed onto the specimen is lost to evaporation during the unmasking operation. In some embodiments, less than about 2% of the predetermined amount of one or more fluids and / or reagents dispensed onto the specimen is lost to evaporation during the unmasking operation. In some embodiments, after the chamber is sealed, substantially no additional fluids and / or reagents are dispensed onto the substrate. In some embodiments, after the chamber is sealed, no additional fluids and / or reagents are dispensed onto the substrate.

[0075] In some embodiments, the method further comprises pre-pressurizing the sealing chamber. In some embodiments, the method further comprises introducing steam into the sealing chamber.

[0076] In some embodiments, the lower plate further includes at least one heating element. In some embodiments, the lower plate further includes at least three heating elements, where a first heating element of the at least three heating elements is disposed below the substrate stage, and a second heating element and a third heating element of the at least three heating elements are disposed adjacent to the first heating element of the at least three heating elements, respectively. In some embodiments, a first temperature gradient is maintained between the first heating element and the second heating element of the at least three heating elements during the unmasking operation, and a second temperature gradient is maintained between the second heating element and the third heating element of the at least three heating elements during the unmasking operation.

[0077] In some embodiments, the force-generating member applies an external force to the sealed chamber. In some embodiments, the force-generating member is selected from the group consisting of a motor, a piston, a spring, a screw mechanism, a lever, and a cam mechanism. In some embodiments, at least one of the lower plate and the upper plate is in thermal communication with the thermal management module. In some embodiments, the unmasking operation includes a temperature increase phase and a temperature maintenance phase. In some embodiments, the temperature increase phase includes heating the specimen at a rate ranging from about 1°C / sec to about 4°C / sec. In some embodiments, the temperature increase phase includes heating the specimen at a rate ranging from about 3°C / sec to about 4°C / sec.

[0078] In some embodiments, the unmasking operation further comprises a temperature ramp-down phase. In some embodiments, the temperature ramp-up phase is shorter than the temperature ramp-down phase. In some embodiments, the temperature ramp-down phase comprises cooling the specimen at a rate ranging from about 0.5°C / sec to about 3°C / sec.

[0079] In some embodiments, the unmasking operation is stopped by opening the chamber without a temperature ramp-down phase. In some embodiments, the unmasking operation is stopped by introducing a predetermined amount of fluid into the chamber. In some embodiments, the predetermined amount of fluid introduced to stop the unmasking operation ranges from about 0.5 mL to about 5 mL.

[0080] In some embodiments, the method further comprises staining the specimen for the presence of one or more biomarkers after the unmasking operation is completed.

[0081] Another aspect of the present disclosure is a method for unmasking a specimen disposed on a substrate, the method comprising: (a) dispensing a predetermined amount of one or more fluids and / or reagents onto at least a portion of the specimen; (b) sealing the specimen within a chamber, the chamber including (i) a substrate stage and a lower plate having a lower engagement surface, and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface, the substrate being supported by the substrate stage in a horizontal position; and (c) performing an unmasking operation within the sealed chamber. In some embodiments, the one or more fluids and / or reagents dispensed onto the specimen are selected from the group consisting of water and buffer. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto the specimen ranges from about 200 μL to about 1000 μL. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto the specimen ranges from about 250 μL to about 500 μL.

[0082] In some embodiments, the lower plate includes one or more independently operable heating and / or cooling elements. In some embodiments, the upper plate includes one or more independently operable heating and / or cooling elements. In some embodiments, both the lower and upper plates include independently operable heating and / or cooling elements. In some embodiments, any of the heating and / or cooling elements in the upper plate can be operated in conjunction with the heating and / or cooling elements in the lower plate. By independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate stage, any portion of the substrate supported by the substrate stage, and / or the chamber can be controlled.

[0083] In some embodiments, the lower plate has a modular design. In some embodiments, the modular lower plate includes a body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the body can be mounted on, engaged with, or coupled to the thermal management module. In some embodiments, the body includes a substrate stage that is part of and integral with the body. In other embodiments, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body with a separable substrate stage. In this manner, the separable substrate stage can be used as a carrier for the substrate. For example, a substrate can be placed on the separable substrate stage, and the substrate and separable substrate stage pair can be picked up together, transported to the body, and deposited on the body. In some embodiments, the substrate and separate substrate stage pair remain together throughout all or at least a portion of the processing steps used to prepare the sample for microscopy, such as, for example, cover slip baking or stain baking. In some embodiments, a user places a substrate holding a sample on a separate substrate stage, inputs the substrate / substrate stage pair into the system, and following sample processing, removes the substrate / substrate stage from the system and then removes the substrate with the processed sample for analysis.

[0084] In some embodiments, the unmasking operation includes heating the specimen disposed on the substrate to a first predetermined temperature for a predetermined period of time. In some embodiments, the first predetermined temperature ranges from about 125°C to about 155°C. In some embodiments, the predetermined duration ranges from about 1 minute to about 10 minutes. In some embodiments, the temperature of the substrate is lower than the temperature of any other components in the chamber during the unmasking operation. In some embodiments, the temperature of the substrate is at least 5°C lower than the temperature of other components in the chamber. In some embodiments, less than about 5% of the predetermined amount of one or more fluids and / or reagents dispensed onto the specimen is lost due to evaporation during the unmasking operation. In some embodiments, no additional fluids and / or reagents are dispensed onto the substrate after the chamber is sealed.

[0085] In some embodiments, the method further includes pre-pressurizing the sealing chamber prior to performing the unmasking operation. In some embodiments, the chamber is pre-pressurized simultaneously with performing the unmasking operation. In some embodiments, the method further includes introducing steam into the sealing chamber.

[0086] In some embodiments, the unmasking operation comprises heating the specimen at a rate ranging from about 1° C. / sec to about 4° C. / sec. In some embodiments, the unmasking operation comprises heating the specimen at a rate ranging from about 3° C. / sec to about 4° C. / sec. In some embodiments, the unmasking operation comprises heating the specimen at a rate ranging from about 0.5° C. / sec to about 2.5° C. / sec.

[0087] Another aspect of the present disclosure is a method for unmasking an analyte disposed on a substrate, the method comprising: (a) dispensing a predetermined amount of one or more fluids and / or reagents onto at least a portion of the analyte; (b) sealing the analyte within a chamber, the chamber including a substrate stage and a lower plate having a lower engagement surface and an upper plate having an upper engagement surface complementary to the lower engagement surface, the substrate being supported by the substrate stage in a horizontal position; and (c) performing an unmasking operation, the unmasking operation including a temperature increase phase, a temperature maintenance phase, and a temperature decrease phase. In some embodiments, the temperature increase phase includes heating the analyte at a rate ranging from about 1° C. / sec to about 4° C. / sec. In some embodiments, the temperature increase phase includes heating the analyte at a rate ranging from about 3° C. / sec to about 4° C. / sec. In some embodiments, the temperature increase phase includes heating the analyte to a predetermined temperature of about 110° C. to about 150° C. In some embodiments, the temperature increasing step comprises heating the specimen to a predetermined temperature of about 120°C to about 145°C.

[0088] In some embodiments, the specimen is maintained at the predetermined temperature for a predetermined period of time ranging from about 1 minute to about 10 minutes. In some embodiments, the specimen is maintained at the predetermined temperature for a predetermined period of time ranging from about 2 minutes to about 7 minutes. In some embodiments, the specimen is maintained at the predetermined temperature for a predetermined period of time ranging from about 3 minutes to about 5 minutes.

[0089] In some embodiments, the temperature ramp-down step comprises cooling the specimen at a rate ranging from about 0.5°C / sec to about 3°C / sec, hi some embodiments, the temperature ramp-down step comprises cooling the specimen at a rate ranging from about 0.5°C / sec to about 2°C / sec.

[0090] In some embodiments, the one or more fluids and / or reagents are selected from the group consisting of water and buffer. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto the specimen ranges from about 200 μL to about 1000 μL. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto the specimen ranges from about 250 μL to about 500 μL.

[0091] Another aspect of the present disclosure is an antigen capture sample prepared by (a) dispensing a predetermined amount of one or more fluids and / or reagents onto at least a portion of the sample; (b) sealing the sample within a chamber, the chamber including a substrate stage and a lower plate having a lower engagement surface, and an upper plate having an upper engagement surface complementary to the lower engagement surface, the substrate being supported by the substrate stage in a horizontal position; and (c) performing an unmasking operation within the sealed chamber.

[0092] Another aspect of the present disclosure is a target collection specimen prepared by (a) dispensing a predetermined amount of one or more fluids and / or reagents onto at least a portion of the specimen; (b) sealing the specimen within a chamber, the chamber including a substrate stage and a lower plate having a lower engagement surface, and an upper plate having an upper engagement surface complementary to the lower engagement surface, the substrate being supported by the substrate stage in a horizontal position; and (c) performing an unmasking operation within the sealed chamber.

[0093] Another aspect of the present disclosure is a target recovery sample prepared by (a) dispensing a predetermined amount of one or more fluids and / or reagents onto a portion of a specimen, (b) sealing the specimen within a chamber, the chamber being formed by contacting a lower engagement surface of a lower plate with an upper engagement surface of an upper plate, the upper engagement surface being complementary to the lower engagement surface, and the lower plate further including a substrate stage for supporting a substrate, and (c) performing an unmasking operation on the specimen within the sealed chamber. In some embodiments, the antigen recovery specimen is a biopsy sample.

[0094] Another aspect of the present disclosure is an antigen capture specimen prepared by (a) dispensing a predetermined amount of one or more fluids and / or reagents onto at least a portion of the specimen, (b) sealing the specimen within a chamber, the chamber including a substrate stage and a lower plate having a lower engagement surface and an upper plate having an upper engagement surface complementary to the lower engagement surface, the substrate being supported by the substrate stage in a horizontal position, and (c) performing an unmasking operation, the unmasking operation including a temperature increase phase, a temperature maintenance phase, and a temperature decrease phase. In some embodiments, the specimen is retained within the sealed chamber as the "coldest" component during all phases of the unmasking operation, e.g., during the temperature increase phase, the temperature maintenance phase, and the temperature decrease phase.

[0095] Another aspect of the present disclosure is a target collection analyte prepared by (a) dispensing a predetermined amount of one or more fluids and / or reagents onto at least a portion of the analyte, (b) sealing the analyte in a chamber, the chamber including a substrate stage and a lower plate having a lower engagement surface and an upper plate having an upper engagement surface complementary to the lower engagement surface, the substrate being supported by the substrate stage in a horizontal position, and (c) performing an unmasking operation, the unmasking operation including a temperature increase phase, a temperature maintenance phase, and a temperature decrease phase. In some embodiments, the analyte is retained in the sealed chamber as the "coldest" component during all phases of the unmasking operation, e.g., during the temperature increase phase, the temperature maintenance phase, and the temperature decrease phase.

[0096] Another aspect of the present disclosure is an unmasked specimen disposed on a substrate, the unmasked specimen being prepared according to a process including: (a) dispensing a predetermined amount of one or more fluids and / or reagents onto a portion of the specimen disposed on the substrate; (b) sealing the specimen within a chamber, the chamber being formed by contacting a lower engagement surface of a lower plate with an upper engagement surface of an upper plate, the upper engagement surface being complementary to the lower engagement surface, and the lower plate further including a substrate stage for supporting the substrate; and (c) performing an unmasking operation on the specimen within the sealed chamber. In some embodiments, the antigen-retrieved specimen is a biopsy sample, and (c) performing an unmasking operation on the specimen. In some embodiments, the unmasked specimen comprises one or more retrieved antigen sites. In some embodiments, the unmasked specimen comprises one or more retrieved nucleic acid targets. In some embodiments, no additional fluids and / or reagents are dispensed onto the substrate after the chamber is sealed.

[0097] In some embodiments, the lower plate includes one or more independently operable heating and / or cooling elements. In some embodiments, the upper plate includes one or more independently operable heating and / or cooling elements. In some embodiments, both the lower and upper plates include independently operable heating and / or cooling elements. In some embodiments, any of the heating and / or cooling elements in the upper plate can be operated in conjunction with the heating and / or cooling elements in the lower plate. By independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate stage, any portion of the substrate supported by the substrate stage, and / or the chamber can be controlled.

[0098] In some embodiments, the lower plate has a modular design. In some embodiments, the modular lower plate includes a body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the body can be mounted on, engaged with, or coupled to the thermal management module. In some embodiments, the body includes a substrate stage that is part of and integral with the body. In other embodiments, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body with a separable substrate stage. In this way, the separable substrate stage can be used as a carrier for the substrate. For example, a substrate can be placed on the separable substrate stage, and the substrate and separable substrate stage pair can be picked up together, transported to the body, and deposited on the body.

[0099] In some embodiments, the substrate and separate substrate stage pair remain together throughout all or at least a portion of the processing steps used to prepare the sample for microscopic analysis, such as, for example, coverslipping or staining. In some embodiments, a user places the substrate bearing the sample on a separate substrate stage, inputs the substrate / substrate stage pair into the system, and following sample processing, removes the substrate / substrate stage from the system and then removes the substrate bearing the processed sample for analysis. In some embodiments, the one or more fluids and / or reagents are selected from the group consisting of water and a buffer having a pH ranging from about 5 to about 10. In some embodiments, the one or more fluids and / or reagents comprise a mixture of deionized water, tris(hydroxymethyl)methylamine, and a chelating agent. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto the specimen ranges from about 200 μL to about 1000 μL. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto the specimen ranges from about 250 μL to about 500 μL.

[0100] In some embodiments, the unmasking operation includes heating the specimen disposed on the substrate to a first predetermined temperature for a predetermined period of time. In some embodiments, the first predetermined temperature is in a range from about 125°C to about 155°C. In some embodiments, the first predetermined temperature is in a range from about 135°C to about 150°C. In some embodiments, the first predetermined temperature is about 140°C. In some embodiments, the predetermined duration is in a range from about 1 minute to about 10 minutes. In some embodiments, the predetermined duration is in a range from about 1 minute to about 5 minutes.

[0101] In some embodiments, the temperature of the substrate is lower than the temperature of any other components in the chamber during the unmasking operation. In some embodiments, the temperature of the substrate is at least 5° C. lower than the temperature of other components in the chamber. In some embodiments, less than about 5% of the predetermined volume of one or more fluids and / or reagents dispensed onto the specimen is lost to evaporation during the unmasking operation. In some embodiments, less than about 2% of the predetermined volume of one or more fluids and / or reagents dispensed onto the specimen is lost to evaporation during the unmasking operation.

[0102] In some embodiments, the sealing chamber is pre-pressurized, hi some embodiments, steam is further introduced into the sealing chamber.

[0103] In some embodiments, the lower plate further includes at least one heating element. In some embodiments, the lower plate further includes at least three heating elements, a first heating element of the at least three heating elements being disposed below the substrate stage, and a second heating element and a third heating element of the at least three heating elements being disposed adjacent to the first heating element of the at least three heating elements. In some embodiments, a first temperature gradient is maintained between the first heating element and the second heating element of the at least three heating elements during the unmasking operation, and a second temperature gradient is maintained between the second heating element and the third heating element of the at least three heating elements during the unmasking operation.

[0104] Another aspect of the present disclosure is an unmasked specimen, the unmasked specimen being prepared in a process in which a specimen disposed on a substrate is held as the coldest component in a sealed chamber during an unmasking operation, the unmasked specimen being prepared by: (a) dispensing a predetermined amount of one or more fluids and / or reagents onto a portion of the specimen disposed on the substrate; (b) placing the specimen-holding substrate containing the dispensed one or more fluids and / or reagents on a substrate stage within a chamber formed from an upper plate and a lower plate, the lower plate including the substrate stage and a lower engagement surface, and the upper plate including an upper engagement surface complementary to the lower engagement surface; (c) sealing the chamber; and (d) performing an unmasking operation on the specimen. In some embodiments, the unmasked specimen comprises one or more recovered antigen sites. In some embodiments, the unmasked specimen comprises one or more recovered nucleic acid targets. In some embodiments, no additional fluids and / or reagents are dispensed onto the substrate after the chamber is sealed.

[0105] Another aspect of the present disclosure relates to a system comprising: (i) at least one chamber, the at least one chamber comprising an upper plate and a lower plate, the lower plate including a lower engagement surface and one or more substrate stages adapted to hold a substrate horizontally within the at least one chamber, the upper plate including an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface, and at least one of the upper plate and the lower plate including at least one of a heating element or a cooling element; and (ii) a dyeing module. In some embodiments, the at least one chamber is a demasking chamber.

[0106] In some embodiments, the at least one unmasking chamber has a predetermined internal volume. In some embodiments, the predetermined internal volume is about 14 cm 3 Approximately 25cm from 3In some embodiments, the predetermined internal volume is in the range of about 16 cm 3 Approximately 22 cm from 3 In some embodiments, the predetermined internal volume is in the range of about 18 cm 3 Approximately 20cm from 3 In some embodiments, the one or more substrate stages are raised relative to the lower engagement surface. In some embodiments, the upper plate includes at least one steam injection port. In some embodiments, the at least one steam injection port is in fluid communication with a steam reservoir or a steam generation element. In some embodiments, the at least one chamber is configured to rapidly heat and / or pressurize a predetermined internal volume. In some embodiments, the lower plate and / or the upper plate may be separated from each other after steam heating and / or steam pressurization.

[0107] In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more alignment members. In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more temperature sensors and / or pressure sensors. In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more temperature sensors in contact with the horizontally held substrate or a fluid disposed thereon. In some embodiments, the one or more temperature sensors directly contact the horizontally held substrate or a fluid disposed thereon.

[0108] In some embodiments, the body of the lower plate and the body of the upper plate both have complementary wedge shapes. In some embodiments, at least one of the lower plate and the upper plate includes at least one seal. In some embodiments, the at least one seal is removable. In some embodiments, the lower plate includes a groove, and the at least one removable seal at least partially engages with the groove. In some embodiments, the at least one removable seal is integrated into a removable seal attachment, and the removable seal attachment engages with a portion of the periphery of the lower plate or the upper plate.

[0109] In some embodiments, at least one of the upper plate or the lower plate is coupled to at least one of a motor, a piston, a spring, a screw mechanism, a lever, or a cam mechanism, hi some embodiments, the upper plate and the lower plate are independently movable.

[0110] In some embodiments, the system further includes a control system. In some embodiments, the control system is adapted to operate at least one heating element or cooling element to uniformly heat and / or cool the one or more substrate stages. In some embodiments, the control system is adapted to operate at least one heating element or cooling element to maintain the one or more substrate stages as the coldest components in the chamber.

[0111] In some embodiments, the system includes at least one substrate transfer device. In some embodiments, the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and a carrier transport. In some embodiments, the system further includes one or more substrate loading stations.

[0112] In some embodiments, the lower plate is modular. In some embodiments, the modular lower plate is transportable to the upper plate. In some embodiments, the upper plate is coupled to a force-generating member. In some embodiments, the force-generating member is configured to shut down when pressure in the chamber exceeds a predetermined threshold.

[0113] Another aspect of the present disclosure is a system comprising: (i) at least one demasking chamber having a predetermined internal volume, the at least one demasking chamber comprising an upper plate and a lower plate, the lower plate comprising a lower engagement surface and one or more substrate stages adapted to hold a substrate horizontally within the at least one demasking chamber, the upper plate comprising an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface, the upper plate comprising one or more steam injection ports for introducing steam into the at least one demasking chamber; (ii) a dyeing module; and (iii) a steam reservoir. In some embodiments, the predetermined internal volume of the at least one demasking chamber is about 14 cm. 3 Approximately 25cm from 3 In some embodiments, the upper and lower plates can be removably coupled to one another (e.g., clamped together) such that upon reaching a predetermined internal pressure and / or temperature, the coupling can be released and the upper and lower plates can be rapidly separated from one another. In some embodiments, at least one of the lower and / or upper plates is in thermal communication with at least one heating element and / or cooling element. In some embodiments, the at least one heating element and / or cooling element can be operated in conjunction with one or more steam injection ports.

[0114] In some embodiments, the top plate is coupled to a force-generating member. In some embodiments, the force-generating member is configured to shut down when pressure in the chamber exceeds a predetermined threshold. In some embodiments, the force-generating member is selected from the group consisting of a motor, a spring, a screw, a lever, a piston, a cam, or any combination thereof.

[0115] In some embodiments, at least one of the bottom plate and / or top plate further comprises one or more alignment members.

[0116] In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more temperature and / or pressure sensors. In some embodiments, at least one of the lower plate and the upper plate further comprises at least one seal. In some embodiments, the at least one seal is removable. In some embodiments, the lower plate comprises a groove, and the at least one removable seal at least partially engages with the groove.

[0117] Another aspect of the present disclosure is a system comprising: (i) at least one specimen processing assembly, the specimen processing assembly comprising: (1) a lower plate comprising a body, the body comprising: (a) a substrate stage having an upper surface adapted to horizontally support a substrate; (b) a lower engagement surface; and (c) a first lower temperature adjustment element in thermal communication with the substrate stage; and (2) an upper plate comprising an upper engagement surface complementary to the lower engagement surface; and (ii) a staining module.

[0118] In some embodiments, the first lower temperature adjustment element is disposed below the substrate stage. In some embodiments, the lower plate further comprises a second lower temperature adjustment element and a third lower temperature adjustment element, each disposed adjacent to the first lower temperature adjustment element. In some embodiments, the first lower temperature adjustment element has a first heat output, the second lower temperature adjustment element has a second heat output, and the third lower temperature adjustment element has a third heat output, the first heat output being smaller than either the second heat output or the third heat output.

[0119] In some embodiments, a temperature gradient is maintained between the first and second lower temperature adjustment elements and between the first and third temperature adjustment elements. In some embodiments, the temperature gradient maintained between the first and second lower temperature adjustment elements is in the range of 2° C. to about 10° C. The temperature gradient maintained between the first and second lower temperature adjustment elements is in the range of 2° C. to about 10° C.

[0120] In some embodiments, the lower plate is modular. In some embodiments, the modular lower plate is transportable to the upper plate. In some embodiments, the upper plate includes an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface. In some embodiments, the upper plate is coupled to a force-generating member. In some embodiments, the force-generating member is configured to shut down when pressure in the chamber exceeds a predetermined threshold.

[0121] In some embodiments, at least one of the upper plate or the lower plate is coupled to at least one of a motor, a piston, a spring, a screw mechanism, a lever, or a cam mechanism.

[0122] In some embodiments, the system further comprises at least one substrate transfer device. In some embodiments, the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and a carrier transport. In some embodiments, the system further comprises one or more substrate loading stations. [Brief explanation of the drawings]

[0123] For a general understanding of the features of the present disclosure, reference is made to the drawings, wherein like reference numerals are used throughout to identify identical elements.

[0124] [Figure 1A] 1 illustrates a top view of a bottom plate according to one embodiment of the present disclosure.

[0125] [Figure 1B] FIG. 1 illustrates a side view of a bottom plate according to one embodiment of the present disclosure.

[0126] [Figure 1C] 1 shows a side view (longitudinal side) of a bottom plate according to one embodiment of the present disclosure.

[0127] [Figure 1D] 1 illustrates a top view of a bottom plate including vacuum ports according to one embodiment of the present disclosure.

[0128] [Figure 1E] 1 illustrates a top view of a bottom plate including vacuum ports according to one embodiment of the present disclosure.

[0129] [Figure 1F] FIG. 1 illustrates a perspective view of a bottom plate including a vacuum port according to one embodiment of the present disclosure.

[0130] [Figure 1G] 1 illustrates a top view of a bottom plate including vacuum ports according to one embodiment of the present disclosure.

[0131] [Figure 1H] 1 illustrates a top view of a bottom plate including vacuum ports according to one embodiment of the present disclosure.

[0132] [Figure 2A] 1 illustrates a top view of a bottom plate according to one embodiment of the present disclosure.

[0133] [Figure 2B] FIG. 1 illustrates a side view of a first end of a bottom plate according to one embodiment of the present disclosure.

[0134] [Figure 2C] FIG. 10 illustrates a side view of a second end of a bottom plate according to one embodiment of the present disclosure.

[0135] [Figure 2D] 1 shows a side view (longitudinal side) of a bottom plate according to one embodiment of the present disclosure.

[0136] [Figure 3A] 1 illustrates a top view of a bottom plate according to one embodiment of the present disclosure.

[0137] [Figure 3B] 1 shows a side view (longitudinal side) of a bottom plate according to one embodiment of the present disclosure.

[0138] [Figure 4A] 1 illustrates a top view of a bottom plate according to one embodiment of the present disclosure.

[0139] [Figure 4B] FIG. 1 illustrates a side view of a first end of a bottom plate according to one embodiment of the present disclosure.

[0140] [Figure 4C] FIG. 10 illustrates a side view of a second end of a bottom plate according to one embodiment of the present disclosure.

[0141] [Figure 4D] 1 shows a side view (longitudinal side) of a bottom plate according to one embodiment of the present disclosure.

[0142] [Figure 5A] 10 illustrates a seal attachment removably coupled to a bottom plate according to one embodiment of the present disclosure.

[0143] [Figure 5B] 10 illustrates a sealing attachment positioned above a bottom plate according to one embodiment of the present disclosure.

[0144] [Figure 6A] 1 illustrates a bottom plate having a wedge shape according to one embodiment of the present disclosure.

[0145] [Figure 6B] 1 illustrates a lower plate having a wedge shape and an upper plate having a wedge shape, the upper and lower plates contacting each other, according to one embodiment of the present disclosure.

[0146] [Figure 6C] FIG. 1 illustrates a side view of a first end of a bottom plate having a wedge shape according to one embodiment of the present disclosure.

[0147] [Figure 6D] FIG. 10 illustrates a side view of a second end of a bottom plate having a wedge shape according to one embodiment of the present disclosure.

[0148] [Figure 6E] 1 illustrates a side view of a bottom plate having a wedge shape according to one embodiment of the present disclosure.

[0149] [Figure 6F] 1 illustrates a side view of a bottom plate having a wedge shape according to one embodiment of the present disclosure.

[0150] [Figure 7A] 10 illustrates a bottom plate coupled to a housing member according to one embodiment of the present disclosure.

[0151] [Figure 7B] 10 illustrates a bottom plate coupled to a housing member according to one embodiment of the present disclosure.

[0152] [Figure 7C] 10 illustrates a bottom plate coupled to a housing member according to one embodiment of the present disclosure.

[0153] [Figure 7D] 10 illustrates a bottom plate coupled to a housing member according to one embodiment of the present disclosure.

[0154] [Figure 8A] 1 illustrates a bottom plate coupled to a heat sink according to one embodiment of the present disclosure.

[0155] [Figure 8B] 1 illustrates a bottom plate coupled to a heat sink according to one embodiment of the present disclosure.

[0156] [Figure 9A] 1 illustrates a top view of a top plate according to one embodiment of the present disclosure.

[0157] [Figure 9B] 1 illustrates a top view of a top plate according to one embodiment of the present disclosure.

[0158] [Figure 9C] FIG. 1 illustrates a side view of a first end of a top plate according to one embodiment of the present disclosure.

[0159] [Figure 9D]FIG. 1 illustrates a side view of a first end of a top plate according to one embodiment of the present disclosure.

[0160] [Figure 9E] FIG. 1 illustrates a side view of a first end of a top plate according to one embodiment of the present disclosure.

[0161] [Figure 9F] FIG. 1 illustrates a side view of a first end of a top plate according to one embodiment of the present disclosure.

[0162] [Figure 9G] 1 illustrates a top plate including a heating element according to one embodiment of the present disclosure.

[0163] [Figure 9H] 1 illustrates a top plate including a heating element according to one embodiment of the present disclosure.

[0164] [Figure 10A] 1 illustrates a bottom plate and a top plate in contact with each other according to one embodiment of the present disclosure.

[0165] [Figure 10B] 1 illustrates a bottom plate and a top plate in contact with each other according to one embodiment of the present disclosure.

[0166] [Figure 10C] 1 illustrates a bottom plate and a top plate in contact with each other according to one embodiment of the present disclosure.

[0167] [Figure 10D] 1 illustrates a bottom plate and a top plate in contact with each other according to one embodiment of the present disclosure.

[0168] [Figure 10E] 1 illustrates a bottom plate and a top plate in contact with each other according to one embodiment of the present disclosure.

[0169] [Figure 10F] 1 illustrates a bottom plate and a top plate in contact with each other according to one embodiment of the present disclosure.

[0170] [Figure 11A] 1 illustrates a bottom plate with an integrated heat sink and a top plate positioned above the bottom plate, according to one embodiment of the present disclosure.

[0171] [Figure 11B] 1 illustrates a bottom plate with an integrated heat sink and a top plate contacting the bottom plate, according to one embodiment of the present disclosure.

[0172] [Figure 12A] 1 illustrates a top view of a bottom plate movably coupled to a rail according to one embodiment of the present disclosure.

[0173] [Figure 12B] FIG. 10 illustrates a side view of a bottom plate movably coupled to a rail, where the rail is horizontally offset by a predetermined amount, according to one embodiment of the present disclosure.

[0174] [Figure 13A] 10 illustrates the relative positions to which the bottom plate can be moved, according to one embodiment of the present disclosure.

[0175] [Figure 13B] 10 illustrates the relative positions to which the bottom plate can be moved, according to one embodiment of the present disclosure.

[0176] [Figure 14A] 10 illustrates the progression of a bottom plate from a first region to a second region and to a third region during processing, according to one embodiment of the present disclosure.

[0177] [Figure 14B]1 illustrates a cam mechanism in communication with an upper plate, according to an embodiment of the present disclosure, which applies pressure to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates.

[0178] [Figure 14C] 1 illustrates a cam mechanism in communication with an upper plate, the cam mechanism adapted to apply a force to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0179] [Figure 14D] FIG. 1 illustrates a side view of an assembly including a force-generating member according to one embodiment of the present disclosure.

[0180] [Figure 14E] 1 illustrates a piston in communication with an upper plate, the piston adapted to apply pressure to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0181] [Figure 14F] 1 illustrates a cam mechanism in communication with an upper plate, the cam mechanism adapted to apply a force to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0182] [Figure 14G] 1 illustrates a piston in communication with an upper plate, the piston adapted to apply pressure to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0183] [Figure 14H]1 illustrates a cam mechanism in communication with an upper plate, the cam mechanism adapted to apply a force to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0184] [Figure 14I] 1 illustrates multiple cam mechanisms joined together via a single camshaft, according to one embodiment of the present disclosure.

[0185] [Figure 14J] 1 illustrates a piston in communication with an upper plate, the piston adapted to apply pressure to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0186] [Figure 14K] 1 illustrates a cam mechanism in communication with an upper plate, the cam mechanism adapted to apply a force to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0187] [Figure 14L] 1 illustrates a piston in communication with an upper plate, the piston adapted to apply pressure to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0188] [Figure 14M] 1 illustrates a cam mechanism in communication with an upper plate, the cam mechanism adapted to apply a force to the upper plate (or structure disposed therebetween) to establish or maintain a sealing engagement between the upper and lower plates, according to an embodiment of the present disclosure.

[0189] [Figure 15A] 1 provides a top view of an assembly including a container in which an unmasking operation can be performed, according to one embodiment.

[0190] [Figure 15B] 1 provides a side view of an assembly including a container and a hinged top plate according to one embodiment of the present disclosure.

[0191] [Figure 15C] 1 provides a perspective view of an assembly including a container in which an unmasking operation can be performed, according to one embodiment.

[0192] [Figure 15D] 1 provides a perspective view of an assembly including a container in which an unmasking operation can be performed, according to one embodiment.

[0193] [Figure 15E] 1 provides a perspective view of an assembly including a container in which an unmasking operation can be performed, according to one embodiment.

[0194] [Figure 16A] 10 illustrates the progression of a bottom plate from a first region to a second region and to a third region during processing, according to one embodiment of the present disclosure.

[0195] [Figure 16B] 10 illustrates the progression of a wedge-shaped bottom plate from a first region to a second region and to a third region during processing, according to one embodiment of the present disclosure.

[0196] [Figure 16C] 10 illustrates the progression of a wedge-shaped bottom plate from a first region to a second region and to a third region during processing, according to one embodiment of the present disclosure.

[0197] [Figure 16D]1 illustrates a wedge-shaped lower plate and upper plate, which can be independently moved toward each other, according to one embodiment of the present disclosure.

[0198] [Figure 17A] 1 illustrates a system having various interoperable modules according to one embodiment of the present disclosure.

[0199] [Figure 17B] 1 illustrates a system having four separate assemblies according to one embodiment of the present disclosure.

[0200] [Figure 17C] 1 illustrates a system having multiple separate assemblies according to one embodiment of the present disclosure.

[0201] [Figure 18A] 1 illustrates an assembly according to one embodiment of the present disclosure.

[0202] [Figure 18B] 1 illustrates an assembly according to one embodiment of the present disclosure.

[0203] [Figure 18C] 1 illustrates an assembly according to one embodiment of the present disclosure.

[0204] [Figure 19A] 1 illustrates a top view of an assembly including a carousel, one or more substrate loaders, and a bottom plate, according to one embodiment of the present disclosure.

[0205] [Figure 19B] FIG. 19B illustrates a side view of the assembly of FIG. 19A including a carousel, one or more substrate loaders, and a bottom plate, according to one embodiment of the present disclosure.

[0206] [Figure 19C]FIG. 19B illustrates a perspective view of the assembly of FIG. 19A including a carousel, one or more substrate loaders, and a bottom plate, according to one embodiment of the present disclosure.

[0207] [Figure 20A] 1 illustrates a perspective view of a system including multiple carousels, substrate holders, substrate loaders, a lower plate, and an upper plate, according to one embodiment of the present disclosure.

[0208] [Figure 20B] 1 illustrates a top view of a system including multiple carousels, substrate holders, substrate loaders, lower plates, and upper plates, according to one embodiment of the present disclosure.

[0209] [Figure 21A] 1 illustrates the placement of heating and / or cooling elements relative to the top and bottom plates according to one embodiment of the present disclosure.

[0210] [Figure 21B] 1 illustrates the placement of heating and / or cooling elements relative to the top and bottom plates according to one embodiment of the present disclosure.

[0211] [Figure 21C] 1 illustrates the placement of heating and / or cooling elements relative to the top and bottom plates according to one embodiment of the present disclosure.

[0212] [Figure 21D] 1 illustrates the placement of heating and / or cooling elements relative to the top and bottom plates according to one embodiment of the present disclosure.

[0213] [Figure 22A] 22C shows a lower plate formed by two substrate stages according to one embodiment of the present disclosure, the lower plate being complementary to the upper plate of FIG. 22B.

[0214] [Figure 22B] 22B shows a top plate configured with two recessed cavities, which is complementary to the bottom plate of FIG. 22A, according to one embodiment of the present disclosure.

[0215] [Figure 23A] 1 illustrates an upper plate and a lower plate adjacent to each other, the upper plate including a deformable sealing element and the lower plate including a tab, according to one embodiment of the present disclosure.

[0216] [Figure 23B] 1 illustrates an upper plate and a lower plate engaged with one another, the upper plate including a deformable sealing element and the lower plate including a tab, according to one embodiment of the present disclosure.

[0217] [Figure 24A] 1 illustrates a side cross-sectional view of a body of a bottom plate including one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0218] [Figure 24B] 1 illustrates a side cross-sectional view of a body of a bottom plate including one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0219] [Figure 24C] 1 shows a cross-sectional side view (longitudinal side of the body) of a lower plate body including one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0220] [Figure 24D] 1 illustrates a side cross-sectional view of a body of a bottom plate including one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0221] [Figure 25A]1 shows a side cross-sectional view of a main body of a lower plate including one or more heating elements and / or cooling elements (e.g., bores into which one or more heating modules, e.g., heating cartridges, can be inserted) according to one embodiment of the present disclosure.

[0222] [Figure 25B] 1 illustrates a side cross-sectional view of a body of a lower plate including one or more heating and / or cooling elements (e.g., one or more fluid channels that function as conduits for fluids that can be heated and / or cooled, such as heated and / or cooled to a predetermined temperature or heated and / or cooled on demand, according to commands from a control unit), according to one embodiment of the present disclosure.

[0223] [Figure 25C] 1 illustrates a side cross-sectional view of a body of a lower plate including one or more heating and / or cooling elements (e.g., bores, fluid channels for inserting one or more heating modules) according to one embodiment of the present disclosure.

[0224] [Figure 25D] 1 illustrates a side cross-sectional view of a body of a bottom plate including one or more heating and / or cooling elements (eg, multiple fluid channels) according to one embodiment of the present disclosure.

[0225] [Figure 25E] 1 illustrates a side cross-sectional view of a body of a bottom plate including one or more heating and / or cooling elements (e.g., thermoelectric modules) according to one embodiment of the present disclosure.

[0226] [Figure 25F] 1 shows a plan view cutaway of the body of the lower plate including one or more heating and / or cooling elements (e.g., bores, fluid channels for inserting one or more heating modules) according to one embodiment of the present disclosure.

[0227] [Figure 25G]1 shows a top cutaway view of the body of a bottom plate including one or more heating and / or cooling elements (e.g., a network of fluid channels) according to one embodiment of the present disclosure.

[0228] [Figure 25H] 1 shows a top cutaway view of the body of a lower plate including one or more heating and / or cooling elements (e.g., two separate networks of fluid channels) according to one embodiment of the present disclosure.

[0229] [Figure 25I] 10 illustrates fluid flow through fluid channels in the bottom plate, according to one embodiment of the present disclosure. [Figure 25J] 10 illustrates fluid flow through fluid channels in the bottom plate, according to one embodiment of the present disclosure.

[0230] [Figure 26A] 1 illustrates a side cross-sectional view of a body in communication with a thermal management module including one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0231] [Figure 26B] 1 illustrates a side cross-sectional view of a body in communication with a thermal management module, both of which include one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0232] [Figure 26C] 1 illustrates a side cross-sectional view of a body in communication with a thermal management module including one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0233] [Figure 26D] 1 illustrates a side cross-sectional view of a body in communication with a thermal management module, both of which include one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0234] [Figure 26E] 1 illustrates a side cross-sectional view of a body in communication with a thermal management module, both of which include one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0235] [Figure 26F] 1 illustrates an analyte processing device including a substrate supported by a thermal management module and a body in thermal communication with one embodiment of the present disclosure.

[0236] [Figure 26G] 1 illustrates an analyte processing device including a substrate supported by a thermal management module and a body in thermal communication with one embodiment of the present disclosure.

[0237] [Figure 27A] 1 illustrates a set of bodies in a carrier according to one embodiment of the present disclosure.

[0238] [Figure 27B] FIG. 1 illustrates a perspective view of a body inserted or embedded within a carrier according to one embodiment of the present disclosure.

[0239] [Figure 27C] FIG. 1 illustrates a perspective view of a body inserted or embedded within a carrier according to one embodiment of the present disclosure.

[0240] [Figure 27D] 1 illustrates a top view of a body inserted or embedded within a carrier according to one embodiment of the present disclosure.

[0241] [Figure 27E] 1 illustrates a side cutaway view of a body inserted or embedded within a carrier according to one embodiment of the present disclosure.

[0242] [Figure 27F]1 illustrates a perspective view of a body inserted or embedded in a carrier in communication with a pick-up member according to one embodiment of the present disclosure.

[0243] [Figure 27G] 1 illustrates a perspective view of a body inserted or embedded within a carrier, the carrier in communication with a pickup member, and the body and carrier in thermal communication with a thermal management module, according to one embodiment of the present disclosure.

[0244] [Figure 27H] 1 illustrates a perspective view of a body inserted or embedded within a carrier, the carrier being in communication with a pickup member, and the body and carrier being positioned on a thermal management module, according to one embodiment of the present disclosure.

[0245] [Figure 27I] 10 illustrates a forklift device moving a carrier including a body to a thermal management module according to one embodiment of the present disclosure.

[0246] [Figure 28A] 1 illustrates a top view of a body of a bottom plate according to one embodiment of the present disclosure.

[0247] [Figure 28B] 1 illustrates a top view of a body of a bottom plate according to one embodiment of the present disclosure.

[0248] [Figure 28C] FIG. 1 illustrates a perspective view of a body of a bottom plate including vacuum ports and / or one or more recesses according to one embodiment of the present disclosure.

[0249] [Figure 28D] 10 illustrates a body of a bottom plate including one or more recesses according to one embodiment of the present disclosure. This figure also illustrates a gripper device positioned near a substrate.

[0250] [Figure 28E] 10 illustrates a body of a bottom plate including one or more recesses according to one embodiment of the present disclosure. This figure also illustrates a gripper device positioned near a substrate.

[0251] [Figure 28F] 10 illustrates a body of a bottom plate including one or more recesses according to one embodiment of the present disclosure. This figure also illustrates a gripper device positioned near a substrate.

[0252] [Figure 29A] 1 illustrates a side cross-sectional view of a body of a top plate including one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0253] [Figure 29B] 1 illustrates a side cross-sectional view of a body of a top plate including one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0254] [Figure 29C] 1 illustrates a side cross-sectional view of a main body of a top plate including one or more heating and / or cooling elements (eg, fluid channels) according to one embodiment of the present disclosure.

[0255] [Figure 29D] 1 illustrates a side cross-sectional view of a main body of a top plate including one or more heating and / or cooling elements (e.g., bores, fluid channels for inserting one or more heating modules) according to one embodiment of the present disclosure.

[0256] [Figure 29E] 1 illustrates a side cross-sectional view of a main body of a top plate including one or more heating and / or cooling elements (e.g., bores for inserting one or more heating modules) according to one embodiment of the present disclosure.

[0257] [Figure 29F]1 illustrates a planar cutaway view of the body of a top plate including one or more heating and / or cooling elements (e.g., bores, fluid channels for inserting one or more heating modules) according to one embodiment of the present disclosure.

[0258] [Figure 29G] 1 shows a top cutaway view of the body of a top plate including one or more heating and / or cooling elements (e.g., a network of fluid channels) according to one embodiment of the present disclosure.

[0259] [Figure 30A] 1 shows a side cross-sectional view of a lower plate and an upper plate in communication with each other to form a chamber therebetween, and also shows that the upper and / or lower plates include one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0260] [Figure 30B] 1 shows a cross-sectional side view of a lower plate and an upper plate in communication with each other, forming a chamber therebetween, and also shows that the upper and / or lower plates include one or more heating and / or cooling elements (fluid channels, bores for inserting one or more heating modules) according to one embodiment of the present disclosure.

[0261] [Figure 30C] 1 shows a side cross-sectional view of a lower plate and an upper plate in communication with each other, forming a chamber therebetween, and also shows that the upper and / or lower plates include one or more heating and / or cooling elements (fluid channels), according to one embodiment of the present disclosure.

[0262] [Figure 30D]1 shows a cross-sectional side view of a lower plate and an upper plate in communication with each other, forming a chamber therebetween, and also shows that the upper and / or lower plates include one or more heating and / or cooling elements (fluid channels, thermoelectric modules) according to one embodiment of the present disclosure.

[0263] [Figure 30E] 1 illustrates fluid flow paths between an upper plate and a lower plate, each of which includes one or more fluid channels. [Figure 30F] 1 illustrates fluid flow paths between an upper plate and a lower plate, each of which includes one or more fluid channels.

[0264] [Figure 31A] 1 illustrates a sample processing assembly according to one embodiment of the present disclosure, including a lower plate movably coupled to a subassembly, the lower plate including a lower engagement surface and one or more substrate stages, and an upper plate having an upper engagement surface complementary to the lower engagement surface, and an electrically powered device.

[0265] [Figure 31B] 1 shows a lower plate including a lower engagement surface and one or more substrate stages, a subassembly for transporting the lower plate, an upper plate having an upper engagement surface complementary to the lower engagement surface, and a jack screw mechanism according to one embodiment of the present disclosure.

[0266] [Figure 31C] 1 illustrates a lower plate including a lower engagement surface and one or more substrate stages, a subassembly for transporting the lower plate, an upper plate having an upper engagement surface complementary to the lower engagement surface, and a scissor jack mechanism according to one embodiment of the present disclosure.

[0267] [Figure 31D]1 illustrates a lower plate including a lower engagement surface and one or more substrate stages, a subassembly for transporting the lower plate, an upper plate having an upper engagement surface complementary to the lower engagement surface, and a scissor jack mechanism according to one embodiment of the present disclosure.

[0268] [Figure 31E] 1 illustrates a lower plate including a lower engagement surface and one or more substrate stages, a subassembly for transporting the lower plate, an upper plate having an upper engagement surface complementary to the lower engagement surface, and a scissor jack mechanism according to one embodiment of the present disclosure.

[0269] [Figure 32A] 1 shows a body having one or more attachment members according to one embodiment of the present disclosure.

[0270] [Figure 32B] 1 shows a body with a roughened or patterned surface according to one embodiment of the present disclosure.

[0271] [Figure 32C] 1 shows a body having a coating disposed thereon according to one embodiment of the present disclosure.

[0272] [Figure 33A] 1 illustrates a gripper arm of a gripper device according to one embodiment of the present disclosure.

[0273] [Figure 33B] 1 illustrates a gripper arm of a gripper device according to one embodiment of the present disclosure.

[0274] [Figure 33C] 1 illustrates a gripper arm of a gripper device according to one embodiment of the present disclosure.

[0275] [Figure 33D] 1 illustrates a gripper arm of a gripper device according to one embodiment of the present disclosure.

[0276] [Figure 33E] 1 illustrates a gripper device including a suction cup according to one embodiment of the present disclosure.

[0277] [Figure 34A] 10 illustrates the movement of a substrate with a gripper device to a specimen processing assembly and the subsequent positioning of the substrate on the body of the lower plate of the specimen processing assembly, according to one embodiment of the present disclosure.

[0278] [Figure 34B] 10 illustrates placement of a substrate on a body of a lower plate using a gripper device and gripper arms, according to one embodiment of the present disclosure.

[0279] [Figure 35A] 1 illustrates a forklift apparatus according to one embodiment of the present disclosure.

[0280] [Figure 35B] 1 illustrates a forklift apparatus according to one embodiment of the present disclosure.

[0281] [Figure 35C] 1 illustrates a forklift apparatus according to one embodiment of the present disclosure.

[0282] [Figure 35D] 1 illustrates a forklift apparatus according to one embodiment of the present disclosure.

[0283] [Figure 36A] 1 illustrates a forklift device including a cylinder acting on a top surface of a substrate, according to one embodiment of the present disclosure.

[0284] [Figure 36B] 10 illustrates an alternative view of a forklift device including a cylinder acting on a top surface of a substrate, according to an embodiment of the present disclosure.

[0285] [Figure 36C] 10 illustrates an alternative view of a forklift device including a cylinder acting on a top surface of a substrate, according to an embodiment of the present disclosure.

[0286] [Figure 36D] 10 illustrates an alternative view of a forklift device including a cylinder acting on a top surface of a substrate, according to an embodiment of the present disclosure.

[0287] [Figure 36E] 10 illustrates an alternative view of a forklift device including a cylinder acting on a top surface of a substrate, according to an embodiment of the present disclosure.

[0288] [Figure 36F] 36A-36E illustrate the pick-up of a substrate using the forklift device shown in FIG.

[0289] [Figure 37A] 1 illustrates a gripper device for picking up and moving a carrier according to one embodiment of the present disclosure.

[0290] [Figure 37B] 1 illustrates a gripper device for picking up and moving a carrier according to one embodiment of the present disclosure.

[0291] [Figure 37C] 1 illustrates a gripper device for picking up and moving a carrier according to one embodiment of the present disclosure.

[0292] [Figure 38A] 1 illustrates a substrate loading station having a platform and raised sides according to one embodiment of the present disclosure.

[0293] [Figure 38B] 1 illustrates a substrate loading station having a platform and raised sides according to one embodiment of the present disclosure.

[0294] [Figure 38C] 1 illustrates a substrate loading station having a platform and one or more spring mechanisms according to one embodiment of the present disclosure.

[0295] [Figure 38D] 1 illustrates a substrate loading station having a platform and one or more spring mechanisms according to one embodiment of the present disclosure.

[0296] [Figure 38E] 1 illustrates a substrate loading station having a platform and one or more alignment grippers, according to one embodiment of the present disclosure.

[0297] [Figure 38F] 1 illustrates a substrate loading station having a platform and one or more alignment grippers, according to one embodiment of the present disclosure.

[0298] [Figure 38G] 1 illustrates a substrate loading station having a platform and one or more alignment grippers, according to one embodiment of the present disclosure.

[0299] [Figure 38H] 1 illustrates a substrate loading station having a platform and one or more rollers according to one embodiment of the present disclosure.

[0300] [Figure 39A] 1 illustrates an upper and lower plate communicating with each other to form a chamber according to one embodiment of the present disclosure, which further illustrates four different thermal zones that can be independently adjusted so that at least a portion of the substrate or substrate stage remains relatively the coldest component within the formed chamber.

[0301] [Figure 39B] 1 illustrates an upper and lower plate communicating with each other to form a chamber according to one embodiment of the present disclosure. The figure further illustrates five different thermal zones that can be independently adjusted so that at least a portion of the substrate or substrate stage remains relatively the coldest component within the formed chamber.

[0302] [Figure 39C] 1 illustrates an upper and lower plate communicating with each other to form a chamber according to one embodiment of the present disclosure. The figure further illustrates six different thermal zones that can be independently adjusted so that at least a portion of the substrate or substrate stage remains relatively the coldest component within the formed chamber.

[0303] [Figure 40A] 1 illustrates a plan view of the lower plate showing different thermal zones A, B, B', C, and D. In some embodiments, at least a portion of thermal zone A is in communication with the substrate, and at least a portion of the substrate is maintained at a lower temperature than the temperatures of thermal zones B, B', C, and / or D.

[0304] [Figure 40B] 1 illustrates a side cutaway view of an upper plate in communication with a lower plate, thereby forming a chamber, according to one embodiment of the present disclosure. The view further illustrates thermal zones A, B, C, D, E, and F, which can be independently adjusted such that at least a portion of a substrate disposed within the formed chamber remains at a temperature lower than the temperature of at least thermal zones C, D, E, and F.

[0305] [Figure 40C]1 illustrates a plan view of the upper plate showing different thermal zones E and F. In some embodiments, at least a portion of thermal zone E is in communication with a substrate (such as a substrate disposed within a chamber formed from the upper and lower plates), and at least a portion of the substrate is maintained at a temperature lower than the temperatures of thermal zones E and F.

[0306] [Figure 41A] 1 shows a thermal analysis of an upper and / or lower plate in communication with one or more independently controllable heating and / or cooling elements. [Figure 41B] 1 shows a thermal analysis of an upper and / or lower plate in communication with one or more independently controllable heating and / or cooling elements. [Figure 41C] 1 shows a thermal analysis of an upper and / or lower plate in communication with one or more independently controllable heating and / or cooling elements.

[0307] [Figure 42A] 1 shows the fluid channels in communication with the bottom plate and other system components, allowing the fluid within the fluid channels to be cooled to a predetermined temperature. [Figure 42B] 1 shows the fluid channels in communication with the bottom plate and other system components, allowing the fluid within the fluid channels to be cooled to a predetermined temperature.

[0308] [Figure 43A] 1 provides a flowchart illustrating a method of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure. [Figure 43B] 1 provides a flowchart illustrating a method of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure. [Figure 43C]1 provides a flowchart illustrating a method of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure. [Figure 43D] 1 provides a flowchart illustrating a method of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure. [Figure 43E] 1 provides a flowchart illustrating a method of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure. [Figure 43F] 1 provides a flowchart illustrating a method of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure. [Figure 43G] 1 provides a flowchart illustrating a method of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure. [Figure 43H] 1 provides a flowchart illustrating a method of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure.

[0309] [Figure 44] Examples of times and temperatures used in the temperature ramp-up, temperature hold, and temperature ramp-down steps are provided.

[0310] [Figure 45] 1 provides examples of temperatures generated using a specimen processing assembly according to one embodiment of the present disclosure.

[0311] [Figure 46] A schematic diagram is provided illustrating one or more control systems communicatively coupled to one or more heating elements, one or more cooling elements, and one or more sensors. DETAILED DESCRIPTION OF THE INVENTION

[0312] It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein including multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0313] definition

[0314] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" is defined inclusively, such that "including A or B" means including A, B, or A and B.

[0315] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, e.g., including at least one of a number or list of elements, but including a plurality, and optionally additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (e.g., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0316] Terms such as "comprises," "includes," and "having" are used interchangeably and have the same meaning. Similarly, terms such as "comprises," "includes," and "having" are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprises," and therefore is to be interpreted as open terms meaning "at least the following" and not excluding additional features, limitations, aspects, etc. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus includes at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c. Additionally, while steps and processes may be outlined in a particular order herein, those skilled in the art will recognize that the ordering of steps and processes may vary.

[0317] As used in the specification and claims herein, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements of the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related to those elements specifically identified or not, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally including more than one A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); etc.

[0318] As used herein, the term "antigen" refers to a substance to which an antibody, antibody analog (e.g., aptamer), or antibody fragment binds. Antigens may be endogenous, whereby they are produced intracellularly as a result of normal or abnormal cellular metabolism or due to viral or intracellular bacterial infection. Endogenous antigens include xenogeneic (heterologous), autologous, and idiotypic or allogeneic (homologous) antigens. Antigens may also be tumor-specific antigens or presented by tumor cells. In this case, they are called tumor-specific antigens (TSAs) and generally result from tumor-specific mutations. Antigens may also be tumor-associated antigens (TAAs) presented by tumor and normal cells. Antigens also include CD antigens, which refer to any of several cell surface markers expressed by leukocytes and can be used to distinguish cell lineages or developmental stages. Such markers can be identified by specific monoclonal antibodies and are numbered according to their cluster of differentiation.

[0319] As used herein, the term "fluid" refers to any liquid or liquid composition, including water, solvents, solutions (e.g., polar solvents, non-polar solvents), mixtures, colloids, suspensions, etc., used in specimen processing operations, including adding a liquid or a liquid-containing composition to a substrate, a specimen disposed on a substrate, etc. Fluids may be aqueous or non-aqueous. Non-limiting examples of fluids include solvents and / or solutions for deparaffinizing paraffin-embedded biological specimens, aqueous detergent solutions, washing solutions, rinsing solutions, acidic solutions, alkaline solutions, transport solutions, and hydrocarbons (e.g., alkanes, isoalkanes, and aromatic compounds, such as xylene). Further examples of fluids include solvents (and mixtures thereof) used to dehydrate or rehydrate biological specimens. In some embodiments, washing solutions include a surfactant to facilitate spreading of the washing solution over the specimen-bearing surface of the slide. In some embodiments, an acidic solution includes deionized water, an acid (e.g., acetic acid), and a solvent. In some embodiments, an alkaline solution includes deionized water, a base, and a solvent. In some embodiments, the transfer solution comprises one or more glycol ethers, such as one or more propylene-based glycol ethers (e.g., propylene glycol ether, di(propylene glycol) ether, and tri(propylene glycol) ether), ethylene-based glycol ethers (e.g., ethylene glycol ether, di(ethylene glycol) ether, and tri(ethylene glycol) ether), and functional analogs thereof. Additional cleaning solutions, transfer solutions, acidic solutions, and alkaline solutions, as well as methods of and equipment for applying them, are described in U.S. Patent Application Publication No. 2016 / 0282374, the disclosure of which is incorporated herein by reference in its entirety.

[0320] As used herein, "horizontal" generally refers to an angle within about ±2 degrees of horizontal, e.g., within about ±1 degree of horizontal, e.g., within about ±0.8 degrees of horizontal. Horizontal also refers to a small angle from horizontal, e.g., an angle of about 0.1 to 1.8 degrees from horizontal, e.g., an angle of about 0.2 to 1.2 degrees, e.g., an angle of about 0.3 to 0.8 degrees. For example, a substrate placed or held horizontally has an orientation such that the large surface of the substrate faces approximately up and down and is substantially parallel to the ground. In certain embodiments, a rectangular substrate such as a microscope slide held horizontally has an angle with respect to the horizontal of about 0.0 to 2.0 degrees along its short axis and an angle with respect to the horizontal of about 0.0 to 2.0 degrees along its long axis, again with the large surface of the substrate facing approximately up and down. Similarly, a subassembly (e.g., a rail) placed or positioned horizontally is a subassembly that is substantially parallel to the ground or has an angle within about ±2 degrees of horizontal.

[0321] As used herein, the term "plurality" refers to two or more, such as three or more, four or more, five or more, etc.

[0322] As used herein, the term "reagent" or "rare reagent" refers to a solution or suspension containing a specific binding entity, an antibody (primary antibody, secondary antibody, or antibody conjugate), a nucleic acid probe (an isolated nucleic acid or isolated synthetic oligonucleotide bound to a detectable label or reporter molecule), an unmasking agent (as defined herein), a detection probe, and a solution or suspension of dye or staining molecules (e.g., H&E staining solution, Pap staining solution, etc.). The term "detection probe" refers to a nucleic acid probe or antibody that binds to a specific target (e.g., a nucleic acid sequence, a protein, etc.). A detection probe can contain a detectable label, such as a radioisotope, an enzyme substrate, a cofactor, a ligand, a chemiluminescent or fluorescent agent, a hapten (including, but not limited to, DNP), and an enzyme. Alternatively, a detection probe may not contain a label or tag and may be detected indirectly (e.g., using a secondary antibody specific for the detection probe). The term "antibody conjugate" refers to an antibody conjugated (directly or indirectly) to one or more labels, where the antibody conjugate is specific for a particular target and the label can be detected (directly or indirectly). For example, the antibody conjugate may be bound to a hapten via a polymer linker and / or spacer, and the antibody conjugate may be indirectly detected by the hapten. Alternatively, the antibody conjugate may be coupled to a fluorophore, for example, via a polymer linker and / or spacer, and the antibody conjugate may be directly detected. Examples of antibody conjugates are described in U.S. Patent Application Publication No. 2014 / 0147906, as well as U.S. Pat. Nos. 8,658,389, 8,686,122, 8,618,265, 8,846,320, and 8,445,191, the disclosures of which are incorporated herein by reference in their entireties.

[0323] As used herein, the term "slide" refers to any substrate of any suitable dimensions (e.g., a substrate made entirely or partially of glass, quartz, plastic, silicon, etc.) on which a biological specimen is placed for analysis, more specifically, a "microscope slide" such as a standard 3" x 1" microscope slide or a standard 75mm x 25mm microscope slide. Examples of biological specimens that can be placed on a slide include, but are not limited to, cytological smears, thin tissue sections (such as from a biopsy), and arrays of biological specimens, e.g., tissue arrays, cell arrays, DNA arrays, RNA arrays, protein arrays, or any combination thereof. Thus, in one embodiment, tissue sections, DNA samples, RNA samples, and / or proteins are placed at specific locations on the slide. In some embodiments, the term slide can refer to SELDI and MALDI chips as well as silicon wafers.

[0324] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules that are characterized by binding to each other to the substantial exclusion of binding to other molecules (e.g., a specific binding pair has a binding constant that is at least 10 times higher than the binding constant of either of the two members of the binding pair to other molecules in a biological sample). 3 M -1 Large, 10 4 M -1 Big or 10 5 M -1 (They can have large binding constants.) Examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidins such as streptavidin, and protein A). Specific binding moieties can also include molecules (or portions thereof) that are specifically bound by such specific binding proteins. Specific binding entities include the primary antibodies or nucleic acid probes described above.

[0325] As used herein, the terms "sample," "tissue sample," "specimen," and the like refer to any sample containing biomolecules (such as proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Other examples of organisms include mammals (such as humans, veterinary animals such as cats, dogs, horses, cows, and pigs, and laboratory animals such as mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections or needle biopsies of tissue), cell samples (such as cytological smears, such as Pap smears or blood smears, or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (obtained by lysing cells and separating their components, such as by centrifugation). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucous membranes, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical or needle biopsy), nipple aspirate, earwax, milk, vaginal fluid, saliva, swabs (such as cheek swabs), or any material containing biomolecules derived from an initial biological sample. In certain embodiments, the term "biological sample," as used herein, refers to a sample prepared from a tumor or portion thereof obtained from a subject (such as a homogenized or liquefied sample).

[0326] As used herein, the terms "staining," "staining," and the like generally refer to any treatment of a biological specimen to detect and / or differentiate the presence, location, and / or amount (e.g., concentration) of a specific molecule (e.g., lipid, protein, or nucleic acid) or a specific structure (e.g., normal or malignant cells, cytosol, nucleus, Golgi apparatus, or cytoskeleton) in the biological specimen. For example, staining can provide contrast between a specific molecule or specific cellular structure and the surrounding area of ​​the biological specimen, and the intensity of the staining can provide a measure of the amount of a specific molecule in the specimen. Staining can be used to aid in the observation of molecules, cellular structures, and organisms using not only brightfield microscopes but also other observation tools such as phase contrast microscopes, electron microscopes, and fluorescence microscopes. Some staining performed by the system can be used to visualize cell contours. Other staining performed by the system can rely on the specific cellular component (e.g., molecule or structure) to be stained, with no or relatively little staining of other cellular components. Examples of types of staining methods performed by the system include, but are not limited to, histochemical methods, immunohistochemical methods, and other methods based on reactions between molecules (including non-covalent interactions), such as hybridization reactions between nucleic acid molecules. Staining methods include, but are not limited to, primary staining methods (e.g., H&E staining, Pap staining, etc.), enzyme-linked immunohistochemical methods, and in situ RNA and DNA hybridization methods, such as fluorescent in situ hybridization (FISH).

[0327] As used herein, the term "substantially" refers to the qualitative condition of exhibiting the entire or nearly entire extent or degree of a characteristic or property of interest. In some embodiments, "substantially" means within about 20%. In some embodiments, "substantially" means within about 15%. In some embodiments, "substantially" means within about 10%. In some embodiments, "substantially" means within about 5%.

[0328] As used herein, the term "target" refers to any molecule whose presence, location, and / or concentration is or can be determined. Examples of target molecules include proteins, epitopes, nucleic acid sequences, and haptens, such as haptens covalently bound to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label.

[0329] As used herein, the terms "unmasking" or "unmasking" refer to recovering an antigen or target and / or improving the detection of antigens, amino acids, peptides, proteins, nucleic acids, and / or other targets in fixed tissue. For example, antigenic sites that may otherwise be undetectable could be revealed by, for example, disrupting some of the protein bridges surrounding the antigen during unmasking. In some embodiments, antigens and / or other targets are unmasked by the application of one or more unmasking agents (defined below), heat, and / or pressure. In some embodiments, only one or more unmasking agents are applied to the specimen to achieve unmasking (e.g., no heat or pressure is required). In other embodiments, only heat is applied to achieve unmasking. In some embodiments, unmasking may occur only in the presence of water and additional heat. Unmasking is described in U.S. Patent Application Publication Nos. 2009 / 0170152 and 2009 / 0104654, the disclosures of which are incorporated herein by reference in their entireties. In some embodiments, unmasking may begin once the unmasking agent is applied to the specimen, regardless of when or where the unmasking operation occurs, for example, unmasking may begin once the unmasking agent is applied to the specimen, before the unmasking operation begins.

[0330] As used herein, the term "unmasking operation" refers to an unmasking step or stage that occurs within a sealed chamber. For example, an unmasking operation can include heating and / or pressurizing a specimen disposed on a substrate in the presence of one or more unmasking agents (defined below) within the sealed chamber for a predetermined time. Other steps of an unmasking operation can include cooling the chamber or any portion thereof or depressurizing the sealed chamber. Still other steps of an unmasking operation include quenching, flash boiling, and / or dispensing additional fluids and / or reagents to the specimen while it is within the sealed chamber. In some embodiments, an unmasking operation can include temperature increase, temperature maintenance, temperature decrease, pressure increase, pressure maintenance, and / or pressure decrease stages that occur after the chamber is formed.

[0331] As used herein, the term "unmasking agent" refers to any liquid, including solutions and mixtures, that is dispensed into a specimen to aid in unmasking. In some embodiments, the unmasking agent comprises multiple components, such as those listed below. In some embodiments, the unmasking agent is a buffer. In some embodiments, the buffer has a pH in the range of about 5 to about 10. In other embodiments, the buffer has a pH in the range of about 7 to about 9. In other embodiments, the buffer has a pH in the range of about 7.5 to about 11. Non-limiting examples of buffering agents include citric acid, potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (bicine), N-tris(hydroxymethyl)methylglycine (tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), and combinations thereof. In some embodiments, the unmasking agent is water. In other embodiments, the buffer may be composed of tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (bicine), N-tris(hydroxymethyl)methylglycine (tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or combinations thereof.

[0332] In some embodiments, the unmasking agent comprises a TRIS-based buffer having a basic pH. In some embodiments, the TRIS-based buffer has a pH of about 10 (e.g., at elevated temperatures). In yet other embodiments, the buffer can be a citrate sodium phosphate buffer having a pH of about 6.0 at elevated temperatures. In other embodiments, the unmasking agent comprises about 0.05% citraconic anhydride. In other embodiments, the unmasking agent comprises about 100 mM TRIS and has a pH of about 8 to about 10. In other embodiments, the unmasking agent comprises about 10 mM citrate, about 2 mM EDTA, and about 0.05% Tween 20 and has a pH of about 6.2. In other embodiments, the unmasking agent comprises about 0.01 M citrate buffer and has a pH of about 6.0.

[0333] In some embodiments, the unmasking agent comprises a component that reacts with the released fixative to prevent it from reacting again with the sample. Examples of such unmasking agents include purging agents or dimedone. Alternatively or additionally, the unmasking agent can comprise a component that reversibly reacts with free amino groups on proteins, thus protecting them from reaction with any available formaldehyde, such as citraconic anhydride (CCA).

[0334] In other embodiments, the unmasking agent comprises a chelating agent. Examples of chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), EGTA / AM (EGTA, tetra(acetoxymethyl ester)), (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA), BAPTA / AM (EGTA tetra(acetoxymethyl ester)), 5,5'-dimethyl-BAPTA-AM, 1,2-bis(2-amino-5-methylphenoxy)ethane (MAPTAM), N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), citrate, or an ionophore such as ionomycin or calcimycin, or any combination or mixture thereof.

[0335] In other embodiments, the unmasking agent comprises an enzyme, non-limiting examples of which include proteinases (e.g., trypsin, chymotrypsin, pepsin, or proteinase K), nucleases, glycanases, and hyaluronidases.

[0336] In other embodiments, the unmasking agent comprises a chaotropic agent, non-limiting examples of which include butanol, ethanol, guanidinium salts (e.g., guanidinium hydrochloride or guanidinium thiocyanate), lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, thiourea, and urea.

[0337] In other embodiments, the unmasking agent comprises a nucleophile, e.g., a chemical species that donates an electron pair to an electrophile. Non-limiting examples of nucleophiles include ammonia, primary amines, secondary amines, and tertiary amines. Other examples of nucleophiles include hydrazines, alcohols, and halides.

[0338] In other embodiments, the unmasking agent comprises a Lewis acid. Non-limiting examples of Lewis acids include metal ions such as iron(III) ions, aluminum ions, magnesium ions, or other electron-deficient compounds such as toluenesulfonic acid, boric acid, boron trifluoride, and tartaric acid.

[0339] In other embodiments, the demasking agent may comprise a surfactant. As used herein, "surfactants" are classified as anionic, cationic, or nonionic depending on their chemical mode of action. Generally, surfactants reduce the interfacial tension between two liquids. An example of a surfactant is sodium dodecyl sulfate. Examples of suitable nonionic surfactants include polyethylene glycol monohexadecyl ether, cetostearyl alcohol, cetyl alcohol, cocamide diethanolamine, cocamide monoethanolamine, decyl glucoside, octylphenoxypolyethoxyethanol, polyethylene glycol monoisohexadecyl ether, lauryl glucoside, nonylphenoxypolyethoxyethanol, 4-nonylphenyl-polyethylene glycol, 1-(4-nonylphenyl)-1,4,7,10,13,16,19,22,25 nonaoxaheptacosane-27- ol, nonoxynol, monolaurin, octaethylene glycol monododecyl ether, oleyl alcohol, polyethylene-polypropylene glycol, polyglycerol polyricinoleate, polysorbate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, and polyoxyethylene (20) sorbitan monooleate, octyl-, decyl-, dodecyl-glucopyranoside, -maltoside or deoxycholic acid. Exemplary surfactants are sold under the following names: Brij™ 35, TWEEN™, Tergitol™, Triton™, Ecosurf™, Dowfax™, Polysorbate 80™, BigCHAP, Deoxy BigCHAP, IGEPAL™, Saponin, Thesit™, Nonidet™, Pluronic F-68, digitonin, deoxycholate, etc. Certain disclosed embodiments relate to using a surfactant selected from Brij™ 35, TWEEN™, Tergitol™, Triton™.Additional antigen unmasking agents are disclosed in US Pat. No. 8,486,335, the disclosure of which is incorporated herein by reference in its entirety.

[0340] In some embodiments, the unmasking agent comprises deionized water, TRIS, and a chelating agent. In some embodiments, the unmasking agent comprises deionized water, TRIS, and a chelating agent and has a pH ranging from about 7 to about 9.5. In some embodiments, the unmasking agent comprises deionized water, TRIS, a chelating agent, and a preservative. In some embodiments, the unmasking agent is CC1, available from Ventana Medical Systems, Inc. of Tucson, Arizona, USA. Additional unmasking agents are further described herein and in U.S. Patent Application Publication No. 2009 / 0170152, the disclosure of which is incorporated herein by reference in its entirety.

[0341] overview

[0342] As described herein, the present disclosure relates to a specimen processing assembly including (a) a lower plate and (b) an upper plate complementary to the lower plate. The "complementary" upper and lower plates each include features or sets of features that complement each other. For example, the lower plate can have a first set of features (e.g., a substrate stage and a lower engagement surface), and the upper plate can have a second set of features (e.g., a cavity and an upper engagement surface). In this example, the cavity of the upper plate can be complementary to the substrate stage, and the upper engagement surface can be complementary to the lower engagement surface.

[0343] In some embodiments, the specimen processing assembly is configured such that the lower plate and the upper plate can contact each other to form a chamber, such as a sealed chamber. In some embodiments, one or both of the lower plate and the upper plate are movable. In some embodiments, the lower plate and the upper plate are both independently movable, e.g., independently movable in any of the x, y, and z coordinate directions. In other embodiments, one of the lower plate or the upper plate is movable and the other of the lower plate or the upper plate is fixed. In some embodiments, the lower plate is moved toward the fixed upper plate. In other embodiments, the upper plate is moved toward the fixed lower plate.

[0344] In some embodiments, one or both of the lower plate and / or upper plate include one or more independently operable heating and / or cooling elements. In some embodiments, any of the heating and / or cooling elements in the upper plate can be operated in conjunction with the heating and / or cooling elements in the lower plate. As described further herein, any of the independently operable heating and / or cooling elements in the upper and / or lower plates can also be operated in conjunction with one or more thermal management modules. As a result, by independently controlling the various heating and / or cooling elements present in the upper and lower plates (or in one or more thermal management modules), the temperature of the specimen (e.g., a specimen disposed on a substrate), the substrate, and / or the chamber can be controlled.

[0345] For example, various heating and / or cooling elements present on the upper and lower plates (or those in the thermal management module) can be operated independently such that at least a portion of the substrate or analytes disposed on the substrate remain the coldest structures within the chamber. For example, the substrate or analytes disposed on the substrate can be maintained at a lower temperature than the upper plate, lower plate, ports, valves, chamber walls, and / or any other structures within the chamber formed from the upper and lower plates.

[0346] In some embodiments, one or more independently operable heating and / or cooling elements on either the lower plate and / or the upper plate can be arranged such that their placement and / or positioning during their operation allows for the establishment of one or more temperature gradients within the formed chamber (or along any components of the formed chamber, including the lower and upper plates). In some embodiments, the one or more heating and / or cooling elements are arranged such that during their operation, a temperature gradient is established between the substrate stage and a portion of the chamber surrounding the substrate stage. In this regard, the temperature gradient allows the substrate stage (or at least a portion of the substrate disposed thereon) to be maintained at a relatively lower temperature than other adjacent regions of the chamber. In some embodiments, the temperature gradient is established and maintained during all stages of the unmasking operation (e.g., heating, pressurizing, cooling, depressurizing, quenching, etc.). In some embodiments, the temperature gradient is established and maintained such that a portion of the specimen or substrate is the “coldest” component within the formed chamber. For example, a portion of the specimen and / or substrate can be maintained at a lower temperature than the temperatures of other portions, such as the upper and / or lower plates, chamber walls, nozzles, ports, temperature probes, pressure sensors, etc.

[0347] In some embodiments, the upper plate is coupled to one or more force-generating members. In some embodiments, the one or more force-generating members are selected from a motor, a screw, a lever, a piston, a cam mechanism, or the like. In some embodiments, the one or more force-generating members apply a predetermined amount of force to at least the upper plate or any structure disposed therebetween. In some embodiments, the applied predetermined amount of force is less than the force generated by a predetermined threshold pressure within a chamber formed by the complementary upper and lower plates. In this regard, the predetermined force applied by the force-generating member is less than the force that pushes the upper and lower plates apart, such that the force-generating member can stall or slip, allowing pressure to be released from the formed chamber. Alternatively, the applied predetermined amount of force does not exceed the force generated by the predetermined threshold pressure within the chamber. This may act as a safety mechanism, thus mitigating the buildup of pressure within the chamber that exceeds the predetermined threshold pressure. In some embodiments, the lower and / or upper plate may also include one or more ports and / or valves to allow pressure exceeding the predetermined threshold pressure to be released from the internal environment within the chamber. For example, a spring having a particular spring constant can be used as at least part of the force generating member, so that when the internal pressure of the chamber exceeds a predetermined pressure, the spring can further collapse to release pressure from the chamber.

[0348] The present disclosure also relates to a sample processing assembly including a chamber, such as a chamber formed from a lower plate and a complementary upper plate. In some embodiments, unmasking (e.g., antigen retrieval, target retrieval) can be performed in the chamber. For example, gas and / or steam can be introduced into the chamber to pressurize it. As another example, one or more heating elements can be activated to heat one or more fluids and / or reagents present in the chamber. In some embodiments, one or more cooling elements are activated simultaneously with one or more heating elements to enable uniform heating of the substrate, the sample disposed on the substrate, or the chamber itself. In some embodiments, the chamber can have any size and / or shape. In some embodiments, the chamber is sized to accommodate at least a portion of the substrate and any sample, fluid, and / or reagent disposed thereon.

[0349] The present disclosure also relates to systems including one or more specimen processing assemblies. In some embodiments, the system includes one or more specimen processing assemblies and at least one dispensing device. In some embodiments, the system further includes one or more liquid removal modules, mixing modules, pressurization modules, imaging modules, coverslipping modules, etc. In some embodiments, the system includes a control system having one or more processors and one or more memories.

[0350] Specimen Processing Assembly Components

[0351] As described above, the specimen processing assembly can include a lower plate and an upper plate complementary to the lower plate. In some embodiments, the specimen processing assembly further includes at least one additional component, such as a subassembly, a support member, a substrate loader, a substrate holder, a force-generating member, etc. Each of these components is further described herein.

[0352] Lower Plate

[0353] The specimen processing assembly of the present disclosure includes at least one lower plate, such as that shown in Figures 1-4, or a modular lower plate, such as that shown in Figures 26A-26E.

[0354] 1A-1C, in some embodiments, the lower plate 10 includes a body 16 having a lower engagement surface 11 and one or more substrate stages 12. In some embodiments, the one or more substrate stages 12 each include an upper surface 12C (e.g., an upper planar surface) adapted to support a substrate 15, such as a microscope slide.

[0355] In some embodiments, the one or more substrate stages 12 are raised relative to the lower engagement surface 11. In other embodiments, the one or more substrate stages 12 are recessed relative to the lower engagement surface 11. In still other embodiments, the one or more substrate stages 12 include a first portion that is raised or horizontal relative to the lower engagement surface 11 and a second portion that is recessed or horizontal relative to the lower engagement surface 11.

[0356] In some embodiments, the substrate stage 12 has a substantially rectangular shape (see, e.g., FIG. 1A or 1D). In other embodiments, the substrate stage 12 has rounded edges. In still other embodiments, the substrate stage 12 has a chamfered edge 172 (see, e.g., FIG. 1H). In some embodiments, the substrate stage 12 approximates the size of the substrate. In other embodiments, the substrate stage 12 is larger than the size of the substrate. In still other embodiments, the substrate stage 12 is smaller than the size of the substrate. For example, the size of the substrate stage 12 may be smaller than a standard microscope slide in at least one dimension. In some embodiments, a substrate stage sized smaller than the substrate allows any condensation formed in the chamber formed by the lower plate and the complementary upper plate to form or move below the slide. In some embodiments, at least a portion of one edge of the substrate overhangs one side of the substrate stage.

[0357] In some embodiments, the lower plate 10 is configured to be complementary to the upper plate 30, as described herein (see, e.g., FIG. 6B). In some embodiments, the lower plate 10 is adapted so that the substrate 15 supported by the substrate stage 12 remains horizontal while the lower plate 10 is moved. In this manner, fluids and / or reagents disposed thereon remain on the substrate; for example, they do not migrate to the edge of the substrate or run off the substrate. In some embodiments, the body 16 of the lower plate 10 is monolithic. In other embodiments, the body 16 includes two or more members bonded together. These and other features of the lower plate 10 are described herein.

[0358] In some embodiments, the body 16 of the lower plate 10 can have any size or shape, such as a polygonal or wedge shape. Similarly, the lower engagement surface 11 can have any size or shape, so long as it is complementary to the upper engagement surface 31 of the upper plate 30 described herein (see, e.g., FIG. 9A). In some embodiments, the lower engagement surface 11 of the lower plate 10 may have the same general shape as the lower plate. Although not shown, in some embodiments, the lower plate 10 can have a curved, rather than straight, lower engagement surface, so long as any upper engagement surface of the upper plate is also complementary to the curved lower engagement surface.

[0359] In some embodiments, the body 16 of the lower plate 10 can have a generally rectangular shape, as shown in any of Figures 1, 2, 3, and 4. In other embodiments, referring to Figure 1A, the body 16 of the lower plate 10 includes a length 2 and a width 3 whose general proportions match those of a microscope slide. In embodiments in which the body 16 has a rectangular shape, the plane formed by the upper planar surface 12C of the substrate stage 12 is parallel to the plane formed by the lower engagement surface 11.

[0360] In other embodiments, the body 16 of the lower plate 10 has a wedge shape. For example, as shown in at least FIG. 6A , the side of the body 16 can have a wedge shape that tapers from approximately a first height 4 to approximately a second height 5. FIGS. 6C and 6D show front end views of both ends of the wedge-shaped body 16, respectively. In some embodiments, the wedge-shaped body 16 can further include a lower engagement surface 11 and a substrate stage 12 ( FIG. 6A ). In some embodiments, as shown in FIG. 6A , the substrate stage 12 is configured such that a plane formed by an upper planar surface 12C of the substrate stage 12 (and any substrate 15 supported by the substrate stage 12) intersects with a plane formed by the lower engagement surface 11. In some embodiments, the upper surface 12C of the substrate stage 12 is configured such that a substrate placed thereon is held horizontally; for example, a substrate placed on the upper surface 12C remains parallel to the ground when a lower plate coupled to a subassembly as described herein traverses the subassembly.

[0361] Referring to Figures 6A and 6B, in some embodiments, the substrate stage first end 12A may be raised relative to the lower engagement surface first portion 11A. Meanwhile, the substrate stage second end 12B may be uniform or recessed relative to the lower engagement surface second portion 11B. In some embodiments, referring to Figure 6A, the substrate stage top surface 12C is provided at a height 6 that is the same as, higher than, or lower than height 4. Figure 6E provides an alternative wedge-shaped body 16 in which the substrate stage first end 12A is raised relative to the lower engagement surface first portion 11A. Meanwhile, the substrate stage second end 12B is recessed relative to the lower engagement surface second portion 11B. Figure 6F provides yet another lower plate 10 having a wedge-shaped body 16.

[0362] Although not shown in FIG. 6A , the substrate stage 12 can include one or more alignment members as described further herein. In some embodiments, the one or more alignment members have a height that is less than the height of a substrate disposed on the substrate stage. In other embodiments, the one or more alignment members have a height that is about half the height of the substrate, or less than half the height of the substrate. Additionally, the body 16 of the wedge-shaped lower plate 10 can include a groove, such as a groove that circumscribes the periphery of the substrate stage 12. In some embodiments, the groove is adapted to at least partially engage a seal, as described further herein.

[0363] In some embodiments, the substrate stage 12 includes a vacuum port 170. In some embodiments, the substrate stage 12 includes a vacuum port and one or more vacuum sealing members 171, as shown in FIGS. 1D through 1H. In some embodiments, the vacuum port 170 is plumbed through the body 16 and is in fluid communication with one or more vacuum lines and / or vacuum sources. In some embodiments, the one or more vacuum sealing members 171 have a non-circular shape. In some embodiments, the one or more vacuum sealing members 171 are constructed from a material such as rubber, silicone, or fluorocarbon rubber. In some embodiments, the one or more vacuum sealing members 171 include O-rings. In some embodiments, the substrate stage includes one vacuum sealing member. In other embodiments, the substrate stage includes two or more vacuum sealing members. In still other embodiments, the substrate stage includes three or more vacuum sealing members. In some embodiments, each of the one or more vacuum sealing members is contiguous with one another.

[0364] In some embodiments, the one or more vacuum sealing members 171 are compressible and / or temporarily deformable (e.g., about 5% compressible, about 10% compressible, about 15% compressible, about 20% compressible, about 25% compressible, etc.). In some embodiments, the one or more vacuum sealing members 171 may be in an uncompressed state in which the one or more vacuum sealing members 171 can extend upwardly beyond the surface 12C of the substrate stage 12. In some embodiments, the one or more vacuum sealing members 171 may be in a compressed state in which the one or more vacuum sealing members 171 can be configured to maintain an airtight seal with the backside of the substrate (e.g., the backside of a microscope slide) as the substrate is pulled against the surface 12C of the substrate stage 12 by vacuum drawn through one of the vacuum ports 170.

[0365] In some embodiments, a vacuum can be drawn between the substrate and the substrate stage. For example, a vacuum sufficient to prevent or limit movement of the substrate along the substrate stage can be drawn. The vacuum can be reduced or eliminated to remove the substrate from the substrate stage. Any of the vacuum ports, sealing members, sealing surfaces, vacuum lines, and vacuum sources described in U.S. Pat. No. 9,989,448 (the disclosure of which is incorporated herein by reference in its entirety) can be utilized within the present disclosure. Additionally, any of the methods of operating vacuum ports such as those described in U.S. Pat. No. 9,989,448 can be adapted for use with the lower plate 10 of the present disclosure.

[0366] Referring at least to FIGS. 2A to 2D , in some embodiments, the substrate stage 12 can further include one or more alignment members 14. In some embodiments, the one or more alignment members 14 are adapted to guide the substrate to an appropriate position on the upper surface 12C of the substrate stage 12, for example, a position that is centered within the upper surface 12C of the substrate stage 12. In some embodiments, the substrate stage 12 can include between 1 and 10 alignment members. In some embodiments, the alignment members 14 are protrusions that protrude perpendicularly from the surface of the substrate stage 12. In some embodiments, the protrusions can have any size and shape, such as a cylindrical or polygonal shape. In some embodiments, the alignment members 14 are pins that protrude from the upper surface 12C of the substrate stage 12. In some embodiments, the one or more alignment members 14 are positioned near one or both of the substrate stage ends 12A and 12B. In some embodiments, the one or more alignment members have a height that is less than the height of the substrate. In other embodiments, the one or more alignment members have a height that is approximately half the height of the substrate, or less than half the height of the substrate.

[0367] In the context of a microscope slide supported by the substrate stage 12, one or more alignment members 14 may be positioned on the substrate stage 12 such that they are located at the ends of the microscope slide. For example, one, two, or three alignment members 14 may be positioned on the substrate stage 12 at the labeled end 15A of the microscope slide 15, and another one, two, or three alignment members 14 may be positioned on the substrate stage 12 at the opposing end of the slide 15 (e.g., the end that holds the specimen). In some embodiments, one or more alignment members 14 may be positioned along the periphery of the longitudinal length of the substrate stage 12. As another example, also in the context of a microscope slide, one or more alignment members 14 may be positioned on the substrate stage 12 along each longitudinal edge of the slide supported by the substrate stage 12. In some embodiments, the alignment members 14 may be equally spaced from one another. In other embodiments, the alignment members 14 may be randomly spaced.

[0368] Referring at least to FIGS. 3A and 3B, in some embodiments, the lower plate 10 can further include a groove 13 at least partially surrounding the body 16 of the lower plate 10. As shown in FIGS. 3A and 4A, in some embodiments, the groove 13 can circumscribe the substrate stage 12 such that the lower plate 10 includes a first lower engagement surface 11A and a second lower engagement surface 11B. In some embodiments, the groove 13 is configured to allow a seal to removably engage with a groove wall (not shown). In some embodiments, a seal inserted into the groove 13 can have a first portion that at least partially engages the groove wall and a second portion that protrudes from the groove 13 and extends at least partially above the engagement surfaces 11A and / or 11B (so as to allow communication with the upper engagement surface 31). In some embodiments, the seal is in the form of an O-ring seal. The term "O-ring seal" refers to an annular seal regardless of its cross-sectional shape. In some embodiments, the substrate stage 12 can further include one or more alignment members, as shown in FIGS. 4A to 4C.

[0369] In some embodiments, the seal may be constructed from a material that is chemically and / or heat resistant. In some embodiments, the seal is constructed from metal. In some embodiments, the seal is compressible and / or temporarily deformable. In some embodiments, the seal is constructed from a material such as rubber, silicone, or fluorocarbon rubber. Suitable examples of rubber include, but are not limited to, ethylene propylene diene monomer (EPDM), ethylene propylene rubber, chloroprene rubber (CR), butyl rubber (IIR), and silicone rubber. Suitable examples of fluororubbers include, but are not limited to, binary vinylidene fluoride rubbers such as vinylidene fluoride / hexafluoropropylene copolymer, vinylidene fluoride / trifluorochloroethylene copolymer, and vinylidene fluoride / pentafluoropropylene copolymer; ternary vinylidene fluoride rubbers such as vinylidene fluoride / tetrafluoroethylene / hexafluoropropylene copolymer, vinylidene fluoride / tetrafluoroethylene / perfluoroalkyl vinyl ether copolymer, and vinylidene fluoride / tetrafluoroethylene / propylene copolymer; tetrafluoroethylene / propylene copolymer; tetrafluoroethylene / perfluoroalkyl vinyl ether copolymer; and thermoplastic fluororubbers.

[0370] In other embodiments, the seal may be composed of a perfluoroelastomer such as those described in U.S. Patent No. 7,834,096, the disclosure of which is incorporated herein by reference in its entirety. In still other embodiments, the seal may be composed of nitriles such as acrylonitrile-butadiene (NBR), hydrogenated nitrile (HNBR), and carboxylated nitrile (XNBR), fluorocarbons (FKM), fluorosilicones (FVMQ), perfluoroelastomers (FFKM), tetrafluoroethylene-propylene (FEPM), ethylene acrylate (AEM), polyacrylates, and thermoplastic polyurethanes. In further embodiments, the seal may be composed of a heat-resistant material such as any of those described in U.S. Patent No. 7,919,554, the disclosure of which is incorporated herein by reference in its entirety.

[0371] In some embodiments, the seal 18 is integrated into the removable seal attachment 17, as shown in FIG. 5B . In some embodiments, the removable seal attachment 17 is configured to engage with a portion of the lower plate 10 (although the removable seal attachment 17 can also be configured to engage with a portion of the upper plate 30). For example, the removable seal attachment 17 can include one or more clips 24 that facilitate attachment to a portion of the lower plate 10 (see, e.g., FIG. 5B ). In other embodiments, the removable seal attachment 17 can frictionally engage the periphery of the lower plate 10. In yet other embodiments, the removable seal attachment 17 can be threaded into the lower plate 10. In some embodiments, the seal 18 of the removable seal attachment 17 fits into the groove 13 of the lower body and at least partially engages with the walls of the groove 13. In some embodiments, at least a portion of the seal 18 of the removable seal attachment 17 extends above the top surface of the removable seal attachment 17. In some embodiments, the seal is positioned so that it can contact a portion of the upper engagement surface 31. In some embodiments, the removable seal attachment 17 includes an upper surface 25 that serves as an engagement surface, such as a lower engagement surface, to facilitate contact with the upper engagement surface 31 of the upper plate 30 and / or formation of a seal. In some embodiments, the removable seal attachment 17 serves to retain the seal formed between the upper and lower plates, particularly when the chamber formed from the upper and lower plates is pressurized. In some embodiments, the removable seal attachment 17 includes a cavity 26 so that the substrate stage 12 (and any substrate 15 supported by the substrate stage 12) can be exposed for further processing when the removable seal attachment 17 is coupled to the lower plate 10.

[0372] In some embodiments, the lower plate 10 includes one or more recesses in the body 16 adjacent to the substrate stage 12. For example, FIGS. 28A, 28B, and 28C each show the body 16 having two recesses 173A and 173B, one adjacent to each longitudinal side of the substrate stage 12. The recesses 173A and 173B can be any size and / or shape. Furthermore, the recesses 173A and 173B can have any depth within the body 16. In some embodiments, the two recesses 173A and 173B are aligned with each other. In other embodiments, the two recesses 173A and 173B are offset from each other. In some embodiments, recesses are also provided within the substrate stage 12 itself.

[0373] In some embodiments, one or more recesses are sized to accommodate a gripper device, e.g., to accommodate arms of a gripper device adapted to pick up and move a substrate. Suitable gripper devices for picking up slides supported by a substrate stage are described herein. Figures 28D and 28E show the body 16 of the bottom plate 10 including one or more recesses 173A and 173B, and further show a gripper device 460 having cutout ends 462A and 462B in the gripper arms 461A and 461B, respectively. These figures show the cutout ends 462A and 462B inserted into the recesses 173A and 173B in the body 16. In the configuration shown in Figure 28E, the gripper arms 461A and 461B are in an extended configuration, but the gripper arms 461A and 461B can be retracted so that the gripper arms 461A and 461B move in the directions X and X' as shown. As gripper arms 461A and 461B are retracted, notched ends 462A and 462B of gripper 460 move toward substrate 15 and at least partially surround the edge of substrate 15, allowing it to be picked up by gripper device 460 and moved, such as in the Z direction as shown. In some embodiments, recesses 173A and 173B in body 16 are sized to accommodate at least notched ends 462A and 462B in both the extended and retracted positions of gripper arms 461A and 461B.

[0374] The lower plate can include one or more heating and / or cooling elements 21. In some embodiments, the one or more heating and / or cooling elements 21 are embedded within the body 16 of the lower plate 10. For example, FIGS. 24A and 24C each show a lower plate 10 having a body 16, thereby showing one or more heating and / or cooling elements 21 embedded within the body 16 of the lower plate 10. Any number of heating and / or cooling elements may be embedded within the body 16 of the lower plate, e.g., one or more heating and / or cooling elements, two or more heating and / or cooling elements, three or more heating and / or cooling elements, four or more heating and / or cooling elements, five or more heating and / or cooling elements, six or more heating and / or cooling elements, etc. Similarly, the body 16 of the lower plate 10 can include any combination of heating and / or cooling elements, such as, for example, one heating element and / or one cooling element, two heating elements, two cooling elements, two heating elements and one cooling element, two cooling elements and one heating element, two cooling elements and two heating elements, three heating elements, three cooling elements, three heating elements and one or two cooling elements, three cooling elements and one or two heating elements, three cooling elements and three heating elements, etc.

[0375] In some embodiments, the one or more heating and / or cooling elements 21 can have any arrangement within the body 16 of the lower plate. For illustrative purposes only, FIG. 24D shows one or more heating elements 21A arranged parallel to one or more cooling elements 21B. However, one skilled in the art will understand that the one or more heating and / or cooling elements can be arranged side-by-side (e.g., heating elements 401A and 401B in FIG. 25A ), in a staggered arrangement (e.g., compare heating elements 401A and 401B with heating elements 411A and 411B), evenly spaced, randomly spaced, etc. In some embodiments, the one or more heating and / or cooling elements can be arranged below the substrate stage. In other embodiments, one or more heating and / or cooling elements may be positioned below the substrate stage, with additional heating and / or cooling elements positioned beside the one or more heating and / or cooling elements positioned below the substrate stage, whereby the additional heating and / or cooling elements are adjacent to the one or more heating and / or cooling elements positioned below the substrate stage (see Figures 25C, 25D, 25F).

[0376] In some embodiments, one or more heating and / or cooling elements can be arranged such that, during their operation (e.g., during any heating and / or cooling step, temperature increase step, or temperature decrease step), their placement and / or positioning relative to the body of the lower plate allows a temperature (or thermal) gradient to be established between different portions of the body 16 of the lower plate 10. In some embodiments, the one or more heating and / or cooling elements are arranged such that, during operation, a temperature gradient is established between the substrate stage and portions of the body surrounding the substrate stage. In this regard, the temperature gradient allows the substrate stage (or at least a portion of the substrate disposed thereon) to be maintained at a relatively lower temperature than other adjacent regions of the body 16 of the lower plate 10. For example, with reference to FIG. 40A , the surface 12C of the substrate stage 12 can be maintained at temperature A, and portions of the body 16 of the lower plate 10 surrounding or adjacent to the substrate stage 12 can be maintained at temperatures B, B′, C, and D. Following this particular example, temperature gradients can be established between A and B, between A and B', between A and C, and between A and D. In some embodiments, if temperature A is maintained at a temperature lower than any of temperatures B, B', C, and D, then surface 12C of substrate stage 12 will be relatively the "coldest" part of bottom plate 10.

[0377] In some embodiments, a temperature gradient is established and maintained during a temperature increase phase (e.g., heating) and a temperature decrease phase (e.g., cooling or reducing the amount of heat supplied). In some embodiments, even before the temperature increase phase begins, the temperature of the substrate stage is maintained at a relatively cooler temperature than the surrounding and / or adjacent portions of the body 16. It is believed that establishing and maintaining a temperature gradient even during temperature increase and temperature decrease allows at least a portion of the substrate to be maintained at a relatively cooler temperature than other portions of the body of the lower plate. In this regard, when the lower plate and the upper plate (described herein) are in communication with each other such that a chamber is formed therebetween (e.g., forming a chamber surrounding the substrate and / or substrate stage), at least a portion of the substrate and / or substrate stage is maintained as the “coldest” component within the formed chamber, e.g., cooler than any other portion of the upper plate and / or lower plate, cooler than any nozzles, ports, temperature probes, pressure sensors, etc.

[0378] For example, one or more heating and / or cooling elements can be independently operated such that, during any temperature ramp-up phase, surface 12C of substrate stage 12 is always maintained at a lower temperature than the temperature of the surrounding and / or adjacent areas of body 16. That is, at any particular time during a temperature ramp-up phase, substrate stage 12 is maintained at a lower temperature than the surrounding and / or adjacent portions of body 16, in some embodiments. For example, with reference to FIG. 40A , assuming the temperature ramp-up occurs over a 10-minute period, when a temperature measurement is taken at the 3-minute time point, temperature A is relatively lower than temperatures B, B′, C, and / or D. Similarly, when a second temperature measurement is taken at the 7-minute time point, temperature A is relatively lower than temperatures B, B′, C, and / or D. Continuing with this example, once a predetermined temperature is reached for any one of portions A, B, B′, C, and / or D (e.g., at the 10-minute time point), the temperature measurement of A taken at this time point will be relatively lower than temperatures B, B′, C, and / or D.

[0379] As described in further detail herein, this is believed to allow any substrate (or any portion of the substrate) supported by the substrate stage to have a relatively lower temperature than adjacent portions of the body of the lower plate. In some embodiments, the temperature gradient between the substrate stage and the portion of the body 16 adjacent to the substrate stage ranges from 5° C. to 50° C. In other embodiments, the temperature gradient between the substrate stage and the portion of the body 16 adjacent to the substrate stage ranges from 5° C. to 40° C. In still other embodiments, the temperature gradient between the substrate stage and the portion of the body 16 adjacent to the substrate stage ranges from 5° C. to 30° C. In a further embodiment, the temperature gradient between the substrate stage and the portion of the body 16 adjacent to the substrate stage ranges from 5° C. to 25° C. In yet another embodiment, the temperature gradient between the substrate stage and the portion of the body 16 adjacent to the substrate stage ranges from 5° C. to 20° C. In other embodiments, the temperature gradient between the substrate stage and the portion of the body 16 adjacent to the substrate stage ranges from 5° C. to 15° C. In other embodiments, the temperature gradient between the substrate stage and the portion of the body 16 adjacent to the substrate stage ranges from 5° C. to 10° C.

[0380] The one or more heating and / or cooling elements embedded within the body 16 of the lower plate 10 may be of any type known to those skilled in the art, including any of those described herein. Those skilled in the art will understand that lower plate embodiments including two or more heating and / or cooling elements may utilize the same or different types of heating and / or cooling elements. By way of example only, the body 16 of the lower plate 10 may include one or more fluid channels for circulating fluid to provide heating and / or cooling of the body, and the body 16 may also include one or more bores into which one or more heating modules (e.g., heating cartridges) may be inserted. As another example, the body 16 of the lower plate 10 may include a thermoelectric cooling module enabling cooling to temperatures below room temperature and one or more fluid tubes or channels for circulating a fluid having a temperature above room temperature.

[0381] In some embodiments, one or more heating elements 21 comprise a heating cartridge. Suitable heating cartridges include those described in U.S. Patent Nos. 3,927,301, 4,617,455, 1,882,365, and 1,433,691. In some embodiments, the heating cartridge comprises a heating foil folded around a cylindrical inner core, such as a ceramic inner core completely surrounded by a metal cylinder.

[0382] The body 16 of the lower plate 10 can include one or more bores into which heating modules, e.g., heating cartridges, can be inserted. In some embodiments, any number of bores can be provided in the body 16 of the lower plate 10, such as one bore, two bores, three bores, four bores, six bores, eight bores, twelve bores, etc. The bores can be arranged in any manner within the body 16 of the lower plate. Thus, the body 16 can accommodate one, two, three, four, six, eight, twelve, etc. heating cartridges. For example, FIGS. 25A and 25C show a body 16 having two bores 401A and 401B, respectively, into which heating cartridges can be inserted.

[0383] In some embodiments, one or more bores may be equally spaced from one another. In other embodiments, one or more bores may be randomly spaced. In some embodiments, the bores open onto the longitudinal sides of the lower plate 10 (see, for example, FIG. 1D, which shows the longitudinal side of the lower plate 10). Alternatively, in other embodiments, the bores may open onto the "short sides" of the lower plate, as shown in FIG. 25A. As shown in FIG. 25F, the bores may traverse the entire length of the body 16 of the lower plate (see bore 401B) or may only partially pass through the body 16 of the lower plate 10 (see bores 401A and 401C). The bores themselves can independently have any size and / or diameter.

[0384] In some embodiments, each of the one or more heating cartridges inserted into body 16 may be independently operable. For example, assuming body 16 of bottom plate 10 includes three heating cartridges inserted into each of three different bores, each of the three heating cartridges may be independently operable, e.g., heating cartridges 1 and 3 can be operated at 80% of their power rating, while cartridge 2 is operated at 40% of its power rating.

[0385] In other embodiments, the one or more heating elements 21 embedded within the body 16 comprise ceramic elements having an electrically conductive ceramic material sintered to a shell of an insulating ceramic material. In other embodiments, the one or more heating elements 21 embedded within the body 16 comprise resistive electric heaters. In yet other embodiments, the one or more heating elements 21 embedded within the body 16 comprise induction coils. In further embodiments, the one or more heating elements embedded within the body 16 utilize one of infrared radiation or microwave radiation to heat a substrate or a sample disposed on a substrate. In some embodiments, infrared radiation or microwave radiation is used to heat a fluid, such as a fluid present in a reservoir or a fluid present on a substrate. In these embodiments, the fluid is heated with infrared or microwave radiation to generate vapor. In some embodiments, the generated vapor is used to heat a sample and / or pressurize a chamber formed from a lower plate and a complementary upper plate, as further described herein.

[0386] In other embodiments, the one or more heating elements embedded within body 16 are selected from one or more of a heating foil, a heating wire, or a heating band. Additional embodiments illustrating the use of heating and / or cooling elements in the upper and lower plates are shown in FIGS. 21A-21D. In some embodiments, any of the heating and / or cooling elements in lower plate 10 can be operated in conjunction with the heating and / or cooling elements in upper plate 30 or any thermal management module. Consequently, one skilled in the art will understand that by controlling the various heating and / or cooling elements present in the upper and lower plates and / or thermal management module, the temperature of the substrate stage, any substrate supported by the substrate stage, and / or chamber can be adjusted, for example, to maintain at least a portion of the specimen or at least a portion of the substrate as the coldest component in any chamber formed from the upper and lower plates.

[0387] In some embodiments, one or more heating or cooling elements 21 embedded within body 16 include Peltier elements or include thermoelectric modules 402. Examples of suitable Peltier elements include those described in U.S. Pat. Nos. 4,685,081, 5,028,988, 5,040,381, and 5,079,618, the disclosures of which are incorporated herein by reference in their entireties. By way of example, in FIG. 25E , which shows a thermoelectric module embedded within body 16 of bottom plate 10, the “hot side” of the thermoelectric module is in thermal communication with body 16 of bottom plate 10. Of course, the “cold side” of the thermoelectric module may likewise be in thermal communication with body 16 of bottom plate 10.

[0388] In other embodiments, one or more heating and / or cooling elements 21 embedded within body 16 include one or more fluid channels that function to circulate a fluid within body 16 of bottom plate 10. In some embodiments, the fluid is a heated oil. In other embodiments, the fluid is a mixture, such as an aqueous mixture including a polyol (e.g., polyethylene glycol or polypropylene glycol) and / or an alcohol (e.g., ethanol or methanol).

[0389] The one or more fluid channels embedded within the body 16 of the lower plate may have any configuration and may be independently operable. For example, with reference to FIGS. 25B and 25F, one or more fluid channels 411A and 411B may be arranged as two independent parallel conduits that independently allow suitable fluid flow through the body 16, e.g., through first openings (414A and 414B) on one side of the body 16, through fluid channels 411A and 411B, and out second openings (413A and 413B) on the other side of the body 16. In this particular example, fluid channels 411A and 411B may each be configured for cooling, each for heating, or one may be configured for heating and the other for cooling. Similarly, fluid channels 411A and 411B may both be configured for heating, but may be configured to heat at different temperatures.

[0390] 25D, showing a cross section of the body 16, illustrates yet another example of the body 16 of the bottom plate 10, including multiple fluid channels 411A-411D. In this particular embodiment, the multiple fluid channels 411A-411D may each be an independent fluid channel, may all be interconnected, or some of the fluid channels may be interconnected (e.g., 411B and 411C may be interconnected) and others may not. In some embodiments, some of the fluid channels 411A-411D may be used to circulate fluids to heat the body 16, the substrate stage 12, the substrate 15, or any analytes deposited on the substrate 15, while others of the fluid channels 411A-411D may be used to circulate fluids to cool the body 16, the substrate stage 12, the substrate 15, or any analytes deposited on the substrate 15. In some embodiments, one or more fluid channels are in communication with one or more fluid delivery lines, ports, fluid reservoirs, heating devices, cooling devices, heat exchangers, pumps, and / or valves so that fluid can be supplied to each fluid channel independently.

[0391] Alternatively, the one or more fluid channels can include a network of fluid channels 412, as shown in FIG. 25G. The network of fluid channels 412 can include one or more independently controllable inlets 414, outlets 413, or valves (not shown) to direct circulation along a particular fluid flow path or to provide circulation within only a portion of the network of channels. In some embodiments, the network of fluid channels can be in communication with a fluid reservoir (not shown), which can be heated or cooled as needed. In some embodiments, the bottom plate includes two or more networks of fluid channels. For example, FIG. 25H shows a bottom plate 10 including a first network of fluid channels 412A and a second network of fluid channels 412B. Each of the networks of fluid channels 412A and 412B can be operated independently, e.g., each can contain fluid heated to a different temperature, each can have a different fluid flow rate, etc. 25I and 25J both show a bottom plate 10 having at least one fluid channel 411 disposed therein, the fluid channel communicating with a recirculation pump 601 and a chiller 602. In some embodiments, the fluid channel 411 further communicates with a fluid reservoir.

[0392] In some embodiments, the substrate or the sample disposed on the substrate may be heated by vapor, as described herein. In these embodiments, heating and / or cooling elements in the lower plate can be used for thermal management in conjunction with the introduction of vapor to the specimen disposed on the substrate.

[0393] In some embodiments, the lower plate can include one or more cooling elements. In some embodiments, the one or more cooling elements are active cooling elements. By way of example, the active cooling elements can include one or more fluid channels to facilitate the flow of coolant and thus provide active cooling of the sample, the upper plate, and / or the lower plate, as described above.

[0394] In other embodiments, the one or more cooling elements are passive cooling elements, such as a heat sink. In some embodiments, the bottom plate 10 includes a heat sink. In some embodiments, the heat sink is integral with the bottom plate 10. In some embodiments, the heat sink is formed within the body 16 of the bottom plate. In other embodiments, the heat sink is formed within a housing member 20 that is engageable with the body 16, as described further herein. For example, as shown in FIGS. 8A and 8B , the heat sink 29 may be integral with the housing member 20. In other embodiments, the heat sink may be secured to the bottom bottom plate 10, for example, glued, screwed, or clipped to either the body 16 or the housing member 20. In some embodiments, the heat sink is a liquid-cooled heat sink, or the heat sink includes one or more fans, thermoelectric coolers, or any combination thereof.

[0395] As mentioned above, in embodiments in which the body 16 of the lower plate 10 includes two or more heating and / or cooling elements 21, the two or more heating and / or cooling elements 21 may be the same or different. For example, FIGS. 25C and 25F each show the body 16 of the lower plate 10 including two different types of heating and / or cooling elements. In these particular examples, the body 16 includes two fluid channels 411A and 411B (which may be interconnected and / or independent of each other) and two bores 401A and 401B into which heating cartridges can be inserted. In this particular embodiment, the two fluid channels can be used independently to heat and / or cool the body 16, the substrate stage 12, the substrate 15, or any analytes deposited on the substrate 15.

[0396] In some embodiments, two independently operable fluid channels 411A and 411B can be used to establish a first temperature (e.g., 95°C), and two independently operable heating cartridges in bores 401A and 401B can be used to establish a second temperature (e.g., 140°C). In this manner, a temperature gradient can be established across the portion of body 16 of lower plate 10. Further following the above example, the portion of body 16 below substrate stage 12 can have a relatively lower temperature than the temperature established around body 16 due to the placement of the various heating and / or cooling elements and the temperatures achieved by each of the various heating and / or cooling elements. According to this example, substrate 15 supported by substrate stage 12 can have a relatively lower temperature than the surroundings of the body, also due to the established temperature gradient.

[0397] In some embodiments, the lower plate 10 can include a device for mixing fluids and / or reagents disposed on the surface of the substrate 15. In some embodiments, the device for mixing fluids and / or reagents is a non-contact mixing device. In some embodiments, the device for non-contact mixing of fluids and / or reagents can be incorporated into the body of the lower plate. In other embodiments, the device for non-contact mixing of fluids and / or reagents can be coupled to the body of the lower plate. In some embodiments, the lower plate 10 can include or be in communication with an element capable of providing vibrations to the substrate 15 supported by the substrate stage 12. In other embodiments, the lower plate 10 can include an acoustic wave generator, such as a transducer. In some embodiments, the transducer is a mechanical transducer. In other embodiments, the transducer is a piezoelectric transducer. In some embodiments, the transducer is comprised of a piezoelectric wafer that generates mechanical vibrations. In some embodiments, a surface transducer is used to dispense or mix a fluid volume on the slide. Suitable devices and methods for non-contact mixing are described in PCT Publication WO 2018 / 215844, the disclosure of which is incorporated herein by reference in its entirety.

[0398] In some embodiments, the lower plate 10 (or the body 16 or the housing member 20) is formed by machining or milling a block of solid material. In other embodiments, the lower plate 10 (or the body 16 or the housing member 20) is formed by a 3D printing process. In other embodiments, the lower plate 10, or any portion thereof, is manufactured from a mold. The lower plate 10 can be manufactured from any material. For example, the lower plate 10, or any component thereof, can be made from a metal or alloy, ceramic, glass, glass-epoxy laminate, or plastic (e.g., derived from a polymer, copolymer, or blend of polymers or copolymers). Examples of suitable polymers include, but are not limited to, polyetheretherketone, polyimide, polyetherimide, polytetrafluoroethylene, polysulfone, polyvinylidene difluoride, and polyphenylene sulfide. Examples of suitable metallic materials include aluminum and steel. In some embodiments, if the lower plate is made from a metal, the metal may be coated or uncoated (e.g., coated with a fluoropolymer).

[0399] In some embodiments, the lower plate has a total mass in the range of about 30 grams to 200 grams. In other embodiments, the lower plate has a total mass in the range of about 40 grams to 180 grams. In yet other embodiments, the lower plate has a total mass in the range of about 40 grams to 160 grams. In yet other embodiments, the lower plate has a total mass in the range of about 40 grams to 140 grams. In other embodiments, the lower plate has a total mass in the range of about 40 grams to 120 grams. In other embodiments, the lower plate has a total mass in the range of about 50 grams to 120 grams. In other embodiments, the lower plate has a total mass in the range of about 50 grams to 100 grams.

[0400] In some embodiments, the lower plate has a heat capacity in the range of about 40 J / C to about 110 J / C. In some embodiments, the lower plate has a heat capacity in the range of about 40 J / C to about 100 J / C. In some embodiments, the lower plate has a heat capacity in the range of about 40 J / C to about 90 J / C. In some embodiments, the lower plate has a heat capacity in the range of about 50 J / C to about 80 J / C. In some embodiments, the lower plate has a heat capacity in the range of about 50 J / C to about 70 J / C. In some embodiments, the lower plate has a heat capacity in the range of about 55 J / C to about 75 J / C.

[0401] Referring to at least FIGS. 7A through 7D, 8A, and 8B, in some embodiments, the lower plate 10 includes a body 16 and a housing member 20. As discussed above, in some embodiments, the body 16 and the housing member 20 of the lower plate 10 are integral with one another and formed from a single monolithic block, e.g., milled or machined from a single monolithic block. In other embodiments, the body 16 of the lower plate 10 is adapted to fit (e.g., releasably fit) within the housing member 20 (see FIGS. 7A through 7D and 8B). In alternative embodiments, the body 16 of the lower plate 10 is fixedly attached to the housing member 20, e.g., glued, screwed, or clamped to the housing member 20. In other embodiments, the body 16 of the lower plate 10 is removably engageable with the housing member 20.

[0402] For example, the body 16 of the lower plate 10 may be inserted into or removed from the housing member 20 as needed. Here, a single type of lower plate 10 can be designed and used with various types of housing members 20, and the various housing members can have different shapes and / or designs (e.g., housing members including an integrated heat sink, housing members including one or more heating and / or cooling elements without an integrated heat sink). In some embodiments, the housing member 20 is configured to receive the body 16 such that the top surface 12C of the substrate stage 12 is recessed relative to the top surface 20C of the housing member 20 ( FIGS. 7A and 8B ).

[0403] In other embodiments, the housing member 20 is configured to receive the body 16 such that the top surface 12C of the substrate stage 12 is flush with the top surface 20C of the housing member 20 ( FIG. 7B ). In yet other embodiments, the housing member 20 is configured to receive the body 16 such that the top surface 12C of the substrate stage 12 is raised relative to the top surface 20C of the housing member 20 ( FIG. 7C ). In a further embodiment, as shown in FIG. 7D , the housing member 20 includes one or more heating elements and / or cooling elements 21 in thermal communication with the body 16, where the one or more heating elements and / or cooling elements 21 may include any combination of those described herein. Again, referring to FIG. 7D , the one or more heating elements and / or cooling elements 21 may be sandwiched between the body 16 of the lower plate 10 and the housing member 20. Alternatively, the one or more heating elements and / or cooling elements may be provided only on one or both of the body 16 of the lower plate 10 and / or the housing member 20.

[0404] In some embodiments, the lower plate 710 has a modular design (see, e.g., FIGS. 26A-26E). A lower plate with a modular design would allow the lower plate (or any portion thereof) to be used in a number of different ways for a number of different purposes in a number of different specimen processing assemblies 100. For example, the modular lower plate 710 may be adapted to mount to, engage with, or couple to other components of the specimen processing assembly 100.

[0405] In some embodiments, the modular bottom plate 710 includes a body 716 having a lower engagement surface 711. In some embodiments, the modular bottom plate 710 includes a thermal management module (described herein). In some embodiments, the body 716 can be mounted to, engaged with, or coupled to the thermal management module 440. For example, FIGS. 26A through 26G each show the body 716 mounted on the thermal management module 440.

[0406] In some embodiments, the body 716 includes a substrate stage 712 that is part of and integral with the body. In some embodiments, the body 716 can be picked up, transferred, and deposited into the thermal management module 440 while the substrate is supported by the substrate stage of the body 716 (the body 716, including the substrate stage 712, and the thermal management module 440 together comprise the modular lower plate 710). In some embodiments, the body 716 may be transferred between different thermal management modules 440 or between different components of the specimen processing assembly 100 or system 200. In some embodiments, the body 716 itself can include any of the components described herein with respect to the body 16 of the lower plate 10, e.g., one or more heating and / or cooling elements, one or more alignment members, etc. The body 716 can be constructed from any material, including any of those described herein with respect to the lower plate 10.

[0407] In other embodiments, the body 716 includes a lower engagement surface but does not include an integrated substrate stage. Rather, the modular lower plate 710 includes the body 716 with a separable substrate stage. In this manner, the separable substrate stage can be used as a carrier for the substrate. For example, a substrate can be placed on the separable substrate stage, and the substrate and separable substrate stage pair can be picked up together, transported to the body 716, and deposited on the body. Continuing this example, once the unmasking operation is completed using the modular lower plate 710 (e.g., including a separate substrate stage, the body 716, and a thermal management module), the separable substrate stage (again functioning as a carrier for the substrate) can be picked up and transported to the specimen processing assembly 100 or other components of the system 200. For example, the separable substrate and separable substrate stage may be transported together to a staining apparatus or a coverslipping apparatus. In some embodiments, the substrate and separate substrate stage pair remain together throughout all processing steps in the system 200.

[0408] 26A , the modular bottom plate 710 includes a substrate stage 712 and a body 716 having a bottom engagement surface 711, the body 716 being in thermal communication with a thermal management module 440. In some embodiments, the thermal management module 440 includes one or more heating and / or cooling elements 21, including any of the heating and cooling elements described herein. For example, the thermal management module 440 can include any number of heating and / or cooling elements, e.g., one heating element, one cooling element, one heating element and one cooling element, two heating elements and one cooling element, two heating elements, two cooling elements, two heating elements and two cooling elements, etc. In some embodiments, the thermal management module 440 can be used to maintain and establish any of the temperature gradients described herein. In some embodiments, both the body 716 and the one or more thermal management modules 440 include one or more heating and / or cooling elements 21 (see, e.g., FIG. 26B ).

[0409] 26C and 26D , the thermal management module 440 includes one or more electrical contacts and / or one or more ports, e.g., one or more electrical contacts for supplying power to another system component and / or one or more ports for supplying fluid to a fluid channel or receiving fluid from a reservoir. Similarly, the body 716 of the modular bottom plate 710 can include one or more complementary electrical contacts for receiving power from the thermal management module 440 and / or one or more complementary ports for receiving fluid from the thermal management module 440.

[0410] For example, as shown in FIG. 26C , the body 716 of the modular bottom plate 710 can include one or more bores 401A and 401B into which heating cartridges are inserted. To maintain the ability for the body 716 to be transportable between different processing regions and / or different specimen processing assemblies 100 (or even other components of the system 200) and moveable without disturbing the electrical wiring connected to the body 716 or its subcomponents, the body 716 can include one or more electrical contacts 442A and 442B so that it can receive power from the thermal management module 440. Similarly, as shown in FIGS. 26C and 26D , the body 716 can include one or more fluid channels 411, and fluid can be received into the fluid channels 411 via one or more ports 441A and 441B, which are complementary to one or more ports on the thermal management module 440. In this manner, the body 716 of the modular bottom plate 710 can be moved without being directly connected to any fluid delivery lines.

[0411] 26E illustrates another embodiment in which the body 716 is in thermal communication with the thermal management module 440, where the body 716 of the modular bottom plate 710 includes bores 401A and 401B into which heating cartridges can be inserted, and the body 716 of the modular bottom plate 710 includes electrical contacts 442A and 442B for supplying power to the heating cartridges inserted in the bores 401A and 401B. The embodiment illustrated in FIG. 26E further illustrates that the thermal management module 440 can include one or more independent fluid channels 411A and 411B. In this particular modular bottom plate 710 configuration, power can be supplied from the thermal management module 440 via the electrical contacts 442A and 442B to the heating cartridges inserted in the bores 401A and 401B to provide heating of the body 716 of the modular bottom plate 710 and / or the substrate stage 712. At the same time, the thermal management module 440 can provide additional heating to the body 716 of the modular bottom plate 710 and / or the substrate stage 712, or can provide cooling to the body 716 of the modular bottom plate 710 and / or the substrate stage 712, depending on the temperature of the fluid circulating in the fluid channels 411A and 411B.

[0412] FIG. 26F shows a substrate 15 disposed on a substrate stage (not shown), which is integral with a body 716. In this particular embodiment, the body 716 is shown in thermal communication with a thermal management module 440 that includes one or more heating and / or cooling elements. FIG. 26F further shows the body 716 in communication with a transport member 433, which can deliver the body 716 to a position above the thermal management module 440 but below the top plate 30. FIG. 26G shows a substrate stage 712 integral with the body 716 of a modular bottom plate 710, which further includes a lower engagement surface 711, a vacuum port 170, and a vacuum sealing element 171. The body 716 of the modular bottom plate 710 is shown supported by and in thermal communication with the thermal management module 440.

[0413] 32A , the body 716 can further include one or more mounting members 431A and 431B disposed at opposite ends (e.g., longitudinal ends) of the body 716. In some embodiments, the one or more mounting members 431A and 431B facilitate movement of the body 716 by one or more grippers or other devices configured to pick up and / or move the body 716, such as in any one of the x, y, and z coordinate directions. For example, a gripper or other pickup device may be configured to pick up and move the body 716 710 by holding it on the first and second mounting members 431A and 431B. In yet other embodiments, the body 716 can include one or more cutouts 430A and 430B in a lower surface of the body 716, which allows a gripper or pickup device (described further herein) to grip the body 716 from underneath and thus support the body during any transport operation (see FIG. 32A ).

[0414] In some embodiments, one or more sides of the body 716 have a roughened or patterned surface 160 to increase friction between one or more sides of the body 716 and a gripper or other pickup device configured to grip the body 716 (see FIG. 32B). In other embodiments, one or more sides of the body 716 have a surface that includes a coating 161, such as a polymer coating, to increase friction between one or more coated sides of the body 716 710 and a gripper or other pickup device configured to grip the body 716 (see FIG. 32C).

[0415] As shown in Figures 27A-27E, the substrate stage 712 and the body 716 having the lower engagement surface 711 may be mounted within a carrier block 432. The carrier block 432 can have any size or shape. In some embodiments, the carrier block has a size and / or shape that facilitates pickup by a gripper device 460 or other pickup device. Figure 27D shows a bottom view of the body 716 mounted within the carrier block 432. Figure 27E shows a side cutaway view of the body 716 mounted within the carrier block 432.

[0416] In some embodiments, the body 716 may be fixed within the carrier block 432. In other embodiments, the body 716 is removably engageable with the carrier block 432. In some embodiments, a single body 716 may be used with carrier blocks 432 of different sizes and / or shapes. In some embodiments, the carrier block 432 is disposable, while the body 716 is reusable. In some embodiments, referring to FIG. 27A , the body 716 further includes one or more attachment members 431A and 431B. In some embodiments, the one or more attachment members 431A and 431B are protrusions that emanate from the body 716 (e.g., from the longitudinal ends of the body) and are integral with the body 716. In some embodiments, the attachment members 431A and 431B on the body 716 can have any size and / or shape, provided that the carrier block 432 has a complementary shape to accommodate the attachment members 431A and 431B. In some embodiments, one or more mounting members 431A and 431B facilitate alignment of body 716 within carrier block 432 and / or secure body 716 within carrier block 432 while the combined body 716 and carrier block 432 is moved, such as moved to thermal management module 440 or other components of specimen processing assembly 100 or system 200.

[0417] In some embodiments, the carrier block 432 is constructed from a material selected from a polymer, a copolymer, a metal, etc. In some embodiments, the carrier block 432 is constructed from the same material as the material of the body 716. In other embodiments, the body 716 is constructed from a first material (e.g., a metal) and the carrier block 432 is constructed from a second material (e.g., a heat-resistant copolymer). In some embodiments, one or more sides of the carrier block 432 have a roughened or patterned surface to increase friction between one or more sides of the carrier block 432 and a gripper or other pickup device. In other embodiments, one or more sides of the carrier block 432 have a surface including a coating, such as a polymer coating, to increase friction between one or more sides of the carrier block 432 and a gripper or other pickup device.

[0418] Movement of the body 716 including the carrier block 432 is illustrated in FIGS. 27F through 27H. Specifically, FIG. 27F illustrates the body 716 including the carrier block 432 in communication with the carrier pickup member 450. In some embodiments, the carrier pickup member 450 includes a carrier pickup body 452 and a pair of carrier pickup arms 451A and 451B. In some embodiments, the carrier pickup body 452 and the carrier pickup arms 451A and 451B are configured to support edges of the carrier block 432. For example, the carrier block 432 can be sized to rest on a portion of the carrier pickup body 452 and both of the carrier pickup arms 451A and 451B. FIG. 27G illustrates the body 716 including the carrier block 432 after the carrier pickup body 450 and the carrier block 432 have been moved to a position adjacent to the thermal management module 440. As illustrated in FIG. 27G, the carrier block 432 is supported by at least the carrier pickup arms 451A and 451B. 27H shows the body 716 including the carrier block 432 after the carrier 432 has been moved via a pickup device (described herein) and positioned above the thermal management module 440. As shown in FIG. 27H, the carrier pickup arms 451A and 451B no longer communicate with, e.g., support, the edge of the carrier block 432. As shown, the carrier pickup device and carrier pickup members can be moved away from the thermal management unit 440 while the carrier block 432 and body 716 remain supported by the thermal management module 440.

[0419] Although not shown, in some embodiments, the lower plate can include two or more substrate stages. In other embodiments, the lower plate can include three or more substrate stages. In yet other embodiments, the lower plate can include four or more substrate stages. In further embodiments, the lower plate can include ten or more substrate stages. By way of example, FIG. 22A shows a lower plate having two substrate stages 12 arranged parallel to one another. In embodiments where the lower plate includes two or more substrate stages, in some embodiments, the upper plate includes features complementary to those of a lower plate having two or more substrate stages (e.g., see FIG. 22B, which includes two cavities 32 adapted to receive at least the substrate 15 of FIG. 22A). In such embodiments, complementary upper and lower plates can be brought together to form a chamber, whereby the chamber is adapted to process two or more substrates together. In some embodiments, the upper and lower plates can be configured such that a divider exists between each substrate stage. In this manner, the divider can act to provide separate processing chambers formed from a single upper plate and a single lower plate. In some embodiments, each of the multiple substrates may be heated by the same heating element, or each may be heated independently by a separate heating element, such as a separate heating element embedded within each substrate stage. Other features of the chamber and how the chamber is formed from a lower plate and an upper plate having certain complementary features are described herein.

[0420] Top Plate

[0421] The specimen processing assembly of the present disclosure includes at least one top plate as shown in Figures 9A through 9H. Referring to Figure 9A, in some embodiments, top plate 30 includes a body 33 having an upper engagement surface 31. In some embodiments, top plate 30 further includes one or more cavities 32 (at least Figure 9A), e.g., one or more cavities recessed within body 33. In some embodiments, top plate can include body 33 having an upper engagement surface 31, but unlike the embodiment shown in Figure 9A, top plate 30 may not include recessed cavities (see, e.g., Figure 10E).

[0422] In some embodiments, the upper plate 30 includes elements that are complementary to features of the lower plate 10. In some embodiments, the features of the upper plate 30 at least partially correspond to features present in the lower plate 10. In particular, the upper plate 30 includes elements that are adapted to match features of the lower plate 10 such that a chamber can be formed when the upper and lower plates are brought together. In this regard, the upper and lower plates may be considered to complement each other, such that the upper plate is adapted to accommodate features of the lower plate (or vice versa).

[0423] Like lower plate 10, upper plate 30 can have any size or shape so long as upper plate 30 is complementary to lower plate 10. For example, as shown in Figures 9A-9H, upper plate 30 can have a generally rectangular shape, whereby both the rectangular upper and lower plates are complementary to each other. Alternatively, as shown in Figures 6A and 6B, upper plate 30 and lower plate 10 can both have a wedge shape, whereby upper plate 30 includes upper engagement surface 31 having a size and / or shape complementary to the size and / or shape of lower engagement surface 11 or 711, but the upper and lower surfaces have different conformations.

[0424] The cavity 32 itself can have any size or shape. In some embodiments, the cavity 32 has a size and / or shape adapted to accommodate features of the lower plate 10, e.g., the substrate stage 12, and any substrate 15 disposed on the substrate stage 12. In this regard, the cavity 32 can have a shape and size complementary to the portion of the lower plate 10 that is raised relative to the lower engagement surface 11 or 711. In some embodiments, the portion of the lower plate 10 that is raised relative to the lower engagement surface 11 may have a first conformation, while the cavity 32 may be adapted to have a second conformation opposite the first conformation (optionally including an additional headspace 40 for accommodating any substrate, fluid, and / or reagent disposed on the substrate). Furthermore, in some embodiments, the cavity 32 may be adapted to accommodate any fluid, reagent, or sample disposed on the surface of the substrate 15 and / or to further accommodate a headspace, e.g., a predetermined dead volume above the substrate surface 15C.

[0425] 9C and 9D provide cross-sectional views of the body 33, showing non-limiting examples of the cavity 32. In some embodiments, the cavity 32 is approximately the same size as or larger than the substrate 15. In some embodiments, the cavity 32 has a volume at least equal to the volume of the substrate (e.g., the volume of the substrate plus a predetermined amount of headspace volume). In some embodiments, the cavity 32 is sized to accommodate not only the substrate but also at least a portion of the substrate stage 12. As a non-limiting example, FIG. 9C shows a cavity 32 having a volume larger than the volume of the substrate 15. For example, the available additional volume can accommodate at least a portion of the substrate stage 12 on which the substrate 15 is supported. As another non-limiting example, FIG. 9D shows a cavity 32 sized to accommodate at least the substrate (e.g., a microscope slide) and any materials disposed on the surface of the substrate, including fluids, reagents, and / or samples. The cavity also includes a headspace 40 having a predetermined volume. 9C-9F show cavity 32 having a monolithic recess, in some embodiments, cavity 32 may be layered, as shown in Figures 10A-10D. In some embodiments, layered cavity 32 can conform to features of bottom plate 10, including those features that are raised relative to bottom engagement surface 11.

[0426] 9B , in some embodiments, the upper plate 30 includes a sealing member 39 that is raised relative to the engagement surface 31, e.g., protruding from the upper engagement surface 31. In some embodiments, the sealing member 39 may be constructed from the same material as the body 33 (e.g., an aluminum body 33 and an aluminum sealing member 39). In some embodiments, the body 33 and the sealing member 39 are machined from a single monolithic block or 3D printed as a single unit. In some embodiments, the sealing member 39 is adapted to fit and / or engage with the walls of the groove 13 in the lower plate 10. Although not shown, in other embodiments, the upper plate can alternatively include a groove similar to the groove 13 in the lower plate 10 to accommodate a seal, e.g., the upper plate can include a groove having walls that can engage with a seal (such as those described herein).

[0427] In some embodiments, the upper plate 30 can include one or more heating elements and / or one or more cooling elements, e.g., one or more elements adapted to heat and / or cool the material of the upper plate. In some embodiments, the one or more heating elements and / or cooling elements can be the same or different from those described for use with the lower plate (see above). In some embodiments, the active cooling elements include multiple fluid channels to facilitate coolant flow and thus provide active cooling of the sample, upper plate, and / or lower plate. In some embodiments, the one or more heating elements and / or cooling elements are integrated or embedded within the upper plate 30 (see, e.g., Figures 9G and 9H). In other embodiments, the one or more heating elements and / or cooling elements are attached externally to one or more surfaces of the body 33 of the upper plate 30. In some embodiments, the upper plate 30 can include an integrated heat sink formed, for example, in the sidewall of the body 33. Additional embodiments illustrating the use of heating elements and / or cooling elements in the upper and lower plates are shown in Figures 21A-21D.

[0428] In some embodiments, the top plate 30 can include one or more heating and / or cooling elements 21 (see FIGS. 29A and 29B). In some embodiments, the one or more heating and / or cooling elements 21 are embedded within the body 33 of the top plate 30. For example, FIGS. 29A and 29C each show a top plate 30 having a body 33, thereby showing one or more heating and / or cooling elements 21 embedded within the body 33 of the top plate 30. Any number of heating and / or cooling elements can be embedded within the body 33 of the top plate 30. Similarly, the body 33 of the top plate 30 may include any combination of heating and / or cooling elements, such as, for example, one heating element and / or one cooling element, two heating elements, two cooling elements, two heating elements and one cooling element, two cooling elements and one heating element, two cooling elements and two heating elements, three heating elements, three cooling elements, three heating elements and one or two cooling elements, three cooling elements and one or two heating elements, three cooling elements and three heating elements, etc.

[0429] In some embodiments, one or more heating and / or cooling elements 21 can have any arrangement within the body 33 of the top plate 30. For illustrative purposes only, FIG. 29B shows one or more heating elements 21A arranged parallel to one or more cooling elements 21B. In some embodiments, one or more heating and / or cooling elements may be arranged side by side (e.g., heating elements 401A and 401B in FIG. 29E), in a staggered arrangement (e.g., compare elements 401A and 401B with elements 411A and 411B as in FIG. 29F), evenly or randomly spaced, etc. In some embodiments, one or more heating and / or cooling elements may be arranged on opposite sides of the cavity 32, with additional heating and / or cooling elements arranged beside one or more heating and / or cooling elements arranged on the opposite side of the cavity 32, the additional heating and / or cooling elements adjacent to one or more heating and / or cooling elements arranged on the opposite side of the cavity (see FIG. 29D).

[0430] The body 33 of the upper plate 30 can include one or more bores into which a heating module, e.g., a heating cartridge, can be inserted. In some embodiments, any number of bores can be provided in the body 33 of the upper plate 30, such as one bore, two bores, three bores, four bores, six bores, eight bores, twelve bores, etc. The bores can be arranged in any manner within the body 16 of the lower plate. Thus, the body 16 can accommodate one, two, three, four, six, eight, twelve, etc. heating cartridges. For example, FIGS. 29D and 29E show a body 33 having two bores 401A and 401B, respectively, into which a heating cartridge can be inserted.

[0431] In some embodiments, one or more bores may be equally spaced from one another. In other embodiments, one or more bores may be randomly spaced. In some embodiments, the bores open onto the longitudinal sides of the upper plate 30. Alternatively, in other embodiments, the bores may open onto the "short sides" of the lower plate, as shown in FIG. 29E. The bores themselves can independently have any size and / or diameter.

[0432] In some embodiments, each of the one or more heating cartridges inserted into body 33 may be independently operable. For example, assuming body 33 of top plate 30 includes three heating cartridges inserted into three bores, each of the three heating cartridges may be independently operable, e.g., heating cartridges 1 and 3 can be operated at 80% of their power rating, and cartridge 2 is operated at 40% of its power rating.

[0433] In other embodiments, one or more heating and / or cooling elements 21 embedded within the body 33 of the top plate 30 include one or more fluid channels that function to circulate a fluid within the body 33 of the top plate 30. In some embodiments, the fluid is a heated oil. In other embodiments, the fluid is a mixture, such as an aqueous mixture including a polyol (e.g., polyethylene glycol or polypropylene glycol) and / or an alcohol (e.g., ethanol or methanol). The one or more fluid channels embedded within the body 33 of the top plate 30 may have any configuration and may be independently operable. For example, with reference to FIGS. 29D and 29F, one or more fluid channels 411A and 411B may be arranged as two independent parallel conduits that independently allow the flow of an appropriate fluid through the body 33. In this particular example, the fluid channels 411A and 411B may each be configured for cooling, each for heating, or one configured for heating and the other for cooling. Similarly, fluid channels 411A and 411B are both configured to heat, but may be configured to heat at different temperatures.

[0434] Alternatively, the one or more fluid channels can comprise a network of fluid channels 412 as shown in Figure 29G. The network of fluid channels 412 can include one or more independently controllable inlets 414, outlets 413, or valves (not shown) to direct circulation along a particular fluid flow path or to provide circulation within only a portion of the network of channels. In some embodiments, the network of fluid channels can be in communication with a fluid reservoir (not shown), which can be heated or cooled as needed.

[0435] In some embodiments, any of the heating and / or cooling elements in the upper plate 30 can be operated in conjunction with the heating and / or cooling elements in the lower plate 10 or any thermal management module. As a result, one skilled in the art will understand that by controlling the various heating and / or cooling elements present in the upper and lower plates and / or thermal management module, the temperature of the substrate stage, any substrate supported by the substrate stage, and / or the chamber can be adjusted. For example, the various heating and / or cooling elements present in the upper and lower plates can be operated such that the substrate, or a specimen disposed on the substrate, remains the coldest structure in the chamber, e.g., the substrate and / or a specimen disposed on the substrate is maintained at a lower temperature than the upper plate, the lower plate, the port, and / or any other structure within the chamber formed from the upper and lower plates. In some embodiments, referring to FIG. 40C , the temperature of the cavity 32 in the upper plate 30 is maintained at a temperature F that is the same as the temperature E of any of the peripheral portions of the body 33 of the upper plate 30. In another embodiment, referring to Figure 40C, the temperature of cavity 32 of top plate 30 is maintained at a temperature F that is lower than the temperature E of any of the surrounding portions of body 33 of top plate 30. In another embodiment, referring to Figure 40C, the temperature of cavity 32 of top plate 30 is maintained at a temperature F that is higher than the temperature E of any of the surrounding portions of body 33 of top plate 30.

[0436] In some embodiments, the top plate can include one or more ports. In some embodiments, the ports include seal or pressure valves so that gas and / or vapor can be released or introduced (e.g., from a chamber formed by complementary top and bottom plates). In some embodiments, the one or more ports are configured to allow gas and / or vapor to be introduced into a chamber formed by the top and bottom plates. In some embodiments, the ports are connected to gas and / or vapor sources, and the valves can be independently operated to allow vapor to be introduced into a chamber formed between the top and bottom plates, for example (see FIG. 21D). In some embodiments, gas and / or vapor is introduced to at least partially heat the substrate and / or sample. In other embodiments, gas and / or vapor is introduced to pre-pressurize a chamber formed by the top and bottom plates. In other embodiments, the one or more ports are configured to introduce one or more fluids and / or reagents.

[0437] In some embodiments, the top plate 30 further includes at least one pressure member. In some embodiments, the at least one pressure member is configured to receive an external force applied to the top plate 30, such as an external force applied by one or more force-generating members. In some embodiments, the one or more force-generating members include a motor, a spring, a screw, a lever, a piston (e.g., a mechanically, electrically, pneumatically, or hydraulically actuated piston), a cam mechanism, or any combination thereof. In some embodiments, the externally applied force is in the range of about 5 Newtons to about 3000 Newtons. In other embodiments, the externally applied force is in the range of about 10 Newtons to about 2000 Newtons. In other embodiments, the externally applied force is in the range of about 10 Newtons to about 1000 Newtons. In other embodiments, the externally applied force is in the range of about 10 Newtons to about 500 Newtons. In still other embodiments, the externally applied force is in the range of about 10 Newtons to about 250 Newtons. In further embodiments, the externally applied force ranges from about 20 Newtons to about 150 Newtons. In some embodiments, the force-generating member applies a predetermined amount of force to the pressure member of the upper plate 30. In some embodiments, the predetermined amount of force applied to the pressure member is either less than a predetermined threshold pressure or does not exceed a predetermined threshold pressure of the internal environment within the chamber formed by the complementary upper and lower plates. In this regard, the force applied by the force-generating member is less than the force pushing the upper and lower plates apart, such that the force-generating member can stall or slide to allow pressure to be released from within the formed chamber.

[0438] In some embodiments, the at least one pressure member is configured to distribute any applied force across the body 33 of the top plate 30, e.g., to distribute the applied force evenly across the top plate 30. In some embodiments, as shown in FIG. 9E, the at least one pressure member includes two parallel bars 34A and 34B. In some embodiments, the parallel bars 34A and 34B extend along the longitudinal sides 41 of the body 33 of the top plate 30 (see also FIGS. 11A and 11B). In other embodiments, with reference to FIG. 9F, the at least one pressure member includes a pressure plate 35, e.g., a monolithic pressure plate coupled to the top surface 33A of the top plate 30. In some embodiments, the pressure plate 35 includes a surface area that is larger than the surface area of ​​the top surface 33A of the top plate 30. In some embodiments, the pressure plate 35 includes a surface area that is smaller than the surface area of ​​the top surface 33A of the top plate 30.

[0439] Although not shown, in some embodiments, the top surface 33A of the body 33 (or, in that case, the pressure plate 35) may include one or more attachment points adapted to couple the top plate to a support member or subassembly.

[0440] 9G and 9H, the pressure plate 35 is adapted to fit within the cavity of the body 33. In some embodiments, the pressure plate 35 adapted to fit within the cavity of the body 33 frictionally engages a raised portion of the body 33 of the top plate 30. In other embodiments, the pressure plate 35 adapted to fit within the cavity of the body 33 is fixedly secured to the body 33 of the top plate 30, for example, glued, screwed, or clamped to the top plate 30. In some embodiments, the heating element 36 is sandwiched between the pressure plate 35 and the body 33. In some embodiments, the pressure plate 35 may further comprise an integral seal 38.

[0441] In some embodiments, the top plate 30 is formed by machining or milling a block of solid material. In other embodiments, the top plate 30 is formed by a 3D printing process. In other embodiments, the top plate 30, or any portion thereof, is manufactured from a mold. The top plate 30 can be manufactured from any material. For example, the top plate 30, or any component thereof, can be made from a metal or alloy, ceramic, glass, or plastic (e.g., derived from a polymer, copolymer, or blend of polymers or copolymers). Examples of suitable polymers include, but are not limited to, polyetheretherketone, polyimide, polyetherimide, polytetrafluoroethylene, polysulfone, polyvinylidene difluoride, and polyphenylene sulfide. Examples of suitable metallic materials include aluminum and steel. In some embodiments, if the top plate is made from a metal, the metal may be coated or uncoated (e.g., coated with a fluoropolymer).

[0442] In some embodiments, the top plate has a total mass in the range of about 30 grams to 200 grams. In other embodiments, the top plate has a total mass in the range of about 40 grams to 180 grams. In yet other embodiments, the top plate has a total mass in the range of about 40 grams to 160 grams. In yet other embodiments, the top plate has a total mass in the range of about 40 grams to 140 grams. In other embodiments, the top plate has a total mass in the range of about 40 grams to 120 grams. In other embodiments, the top plate has a total mass in the range of about 50 grams to 120 grams. In other embodiments, the top plate has a total mass in the range of about 50 grams to 100 grams.

[0443] In some embodiments, the top plate has a heat capacity in the range of about 40 J / C to about 110 J / C. In some embodiments, the top plate has a heat capacity in the range of about 40 J / C to about 100 J / C. In some embodiments, the top plate has a heat capacity in the range of about 40 J / C to about 90 J / C. In some embodiments, the top plate has a heat capacity in the range of about 50 J / C to about 80 J / C. In some embodiments, the top plate has a heat capacity in the range of about 50 J / C to about 70 J / C. In some embodiments, the top plate has a heat capacity in the range of about 55 J / C to about 75 J / C.

[0444] Additional Specimen Processing Assembly Components

[0445] In some embodiments, the specimen processing assembly of the present disclosure includes (i) a lower plate, (ii) an upper plate complementary to the lower plate, and (iii) at least one additional component, hi some embodiments, the at least one additional component is selected from a subassembly, a support member, a force-generating member, a substrate loader, etc.

[0446] In some embodiments, the specimen processing assembly includes one or more subassemblies, such as rails, to which one of the lower plate and / or upper plate can be independently coupled (directly or indirectly). In some embodiments, the one or more subassemblies enable independent movement of the lower plate and / or upper plate in any of the x, y, and z coordinate directions. For example, with reference to at least FIGS. 12A and 12B , the specimen processing assembly of the present disclosure may include a lower plate 10 movably coupled to a subassembly 101 in some embodiments. In some embodiments, the subassembly is a rail. In some embodiments, the subassembly includes a motor coupled to a belt or screw, which may be directly or indirectly coupled to the lower plate 10.

[0447] In some embodiments, the subassembly 101 may be straight or curved, e.g., arcuate, semicircular, etc. In other embodiments, the subassembly 101 may have a complex shape including one or more straight sections and one or more curved sections, e.g., two straight sections with an arc-shaped middle section. In some embodiments, two or more lower plates 10 may be coupled to a single subassembly 101, and each of the two or more plates 10 may move independently or may be coupled to move in tandem.

[0448] In some embodiments, the bottom plate 10 may be coupled directly to the subassembly 101, such as in a track along the top and / or sides of the subassembly 101 (see FIGS. 12A and 12B). In this way, the bottom plate 10 can be slid or moved directly in a track along the subassembly 101, such as along the plane of the subassembly 101.

[0449] In other embodiments, lower plate 10 may be indirectly coupled to subassembly 101. For example, support member 115A may be coupled to (i) a portion of lower plate 10 and (ii) a track of subassembly 101. In this particular embodiment, support member 115A facilitates movement of lower plate 10 along subassembly 101. In some embodiments, support member 115A is configured so that a plane defined by the upper surface of substrate 15 remains horizontal, e.g., parallel to the ground, regardless of movement of lower plate 10 along subassembly 101. In some embodiments, support member 115A includes one or more springs.

[0450] In some embodiments, the subassembly 101 may be positioned horizontally. This is shown in FIG. 12A, where the subassembly 101 is positioned horizontally in the x, y plane. In this embodiment, the horizontal movement of the lower plate 10 along the length of the subassembly 101 is in the x, y plane. FIG. 12A also shows the substrate stage 12 having an upper surface parallel to the ground. Because the rails are positioned parallel to the ground, any substrate 15 supported by the substrate stage 12, as well as any analytes, fluids, and / or reagents disposed thereon, remain parallel to the ground (e.g., in the x, y plane of FIG. 12A) throughout the movement of the lower plate 10.

[0451] In other embodiments, one end of the subassembly 101 may be raised along the z-axis relative to the other end. For example, FIG. 12B shows a subassembly 101 having a first end 113 and a second end 114, where the first end 113 is raised along the z-axis relative to the second end 114 (hereinafter referred to as "offset from horizontal" or "horizontally offset"). In such an embodiment, a component of movement of the lower plate 10 along the horizontally offset subassembly 101 is along the z-axis, e.g., there is a vertical component to the movement. This is further illustrated, for example, in FIGS. 16A and 16B, which show that the lower plate 10 can be moved along the subassembly 101 from end 114 to end 113, and from a first position to a second position, and any intermediate positions therebetween.

[0452] In some embodiments, the horizontal offset can be an angle ranging from about 5 degrees to about 70 degrees. In other embodiments, the horizontal offset can be in the range of about 5 degrees to about 60 degrees. In still other embodiments, the horizontal offset can be in the range of about 5 degrees to about 50 degrees. In still other embodiments, the horizontal offset can be in the range of about 10 degrees to about 50 degrees. In still other embodiments, the horizontal offset can be in the range of about 15 degrees to about 50 degrees. In still other embodiments, the horizontal offset can be in the range of about 20 degrees to about 50 degrees. In still other embodiments, the horizontal offset can be in the range of about 20 degrees to about 45 degrees.

[0453] In embodiments in which the subassembly 101 is offset from horizontal, the support member 115A may be adapted to orient the upper surface of the lower plate 10, and therefore the substrate stage 12, such that any substrate placed thereon remains parallel to the ground and in the x, y plane. In other embodiments, the lower plate itself is configured so that the lower engagement surface remains parallel to the ground, or the surface of the substrate stage remains parallel to the ground.

[0454] In some embodiments, the upper plate 30 may be coupled to the subassembly. In some embodiments, referring to FIG. 14A , the upper plate 30 may be coupled to the subassembly so as to be fixed in space, such as via support members 115B. In embodiments in which the lower plate 10 is fixed, the upper plate may be movably coupled to the subassembly (movable in any of the x, y, and z directions). In these embodiments, the upper plate 30 may be coupled to the subassembly to allow its movement along any of the x, y, and z axes. Alternatively, the upper plate 30 may be coupled to allow its movement only in the z axis.

[0455] In some embodiments, the upper plate may be directly or indirectly coupled to one or more force-generating members. In some embodiments, the one or more force-generating members include a motor, a spring, a screw, a lever, a piston (e.g., a mechanically, pneumatically, electrically, or hydraulically actuated piston), a cam mechanism, or any combination thereof. In some embodiments, the force-generating members facilitate either movement of the upper plate along the z-axis (e.g., to move the upper plate toward the lower plate) or application of a force along the z-axis (such that a force is applied to maintain the upper and lower plates in contact with each other even when a chamber comprising the upper and lower plates is pressurized). In some embodiments, the one or more force-generating members apply a predetermined force to the upper plate. In some embodiments, this predetermined force is limited such that when the pressure in the chamber exceeds a predetermined threshold pressure, the force applied by the force-generating member is overcome, causing the force-generating member to slip or release pressure that may build up in the chamber above the predetermined threshold pressure. For example, if the one or more force-generating members include a spring or piston, the spring or piston may be configured to apply a predetermined amount of force that is less than any force applied when the chamber exceeds the predetermined threshold pressure. In other embodiments, pressure relief ports and / or valves are included in either or both of the bottom and top plates to prevent a predetermined threshold pressure from being exceeded with the chamber.

[0456] In some embodiments, the specimen processing assembly can include a substrate loader 302 so that substrates can be transferred to or removed from the lower plate 10 (see, e.g., FIG. 19A). Additional aspects of substrate loaders and examples of their utilization in particular specimen processing assemblies are described further herein.

[0457] In some embodiments, the specimen processing assembly includes one or more substrate and / or lower plate leveling devices, which can include, for example, one or more adjustable legs or other adjustment protrusions or springs, so that the substrate can be maintained in a substantially horizontal position during processing, as described herein.

[0458] A chamber formed by an upper plate and a lower plate

[0459] As described above, the specimen processing assembly of the present disclosure can include one or more chambers. In some embodiments, each chamber is formed from a lower plate and an upper plate, where the upper plate is complementary to the lower plate. In some embodiments, the chambers formed from the complementary upper plate 30 and lower plate 10, respectively, are adapted to enclose at least the upper surface 15A of the substrate 15 and any fluids, reagents, and / or samples disposed thereon (see, e.g., FIGS. 6B and 10A-10F). In some embodiments, the complementary upper and lower plates may be configured to be disposed within a chamber formed by the substrate or only a portion of the substrate. In other embodiments, the complementary upper and lower plates may be configured to be disposed within a chamber formed by a portion of the substrate and substrate stage.

[0460] As described herein, the upper plate 30 and the lower plate 10 are configured to have complementary sizes and / or shapes such that features present on one of the lower or upper plates are accommodated by the other of the upper or lower plates. In some embodiments, a chamber can be formed when the complementary upper and lower engagement surfaces 31 and 11 (or 711) of the upper and lower plates 30 and 10 (or 710), respectively, contact each other. In some embodiments, the contact between the upper and lower engagement surfaces 31 and 11 (or 711) facilitates the formation of a seal, such as an airtight or moisture-proof seal. In some embodiments, the formation of the seal is further facilitated by applying an external force to one or both of the upper and lower plates, as further described herein.

[0461] In some embodiments, the chambers formed by the lower plate 10 and the upper plate 30, respectively, provide environments that can be controlled according to user preferences. For example, the chambers can be configured such that the environment within or around the formed chambers is different from the environment outside the chambers, e.g., the environment outside the lower and upper plates. In some embodiments, the formed chambers allow analytes, fluids, and / or reagents disposed on the surface of the substrate to be processed at elevated temperatures (e.g., temperatures above room temperature) and / or elevated pressures (e.g., pressures above atmospheric pressure), as further described herein.

[0462] In some embodiments, the chamber formed from the complementary upper and lower plates may be internally pressurized, such as by heating a fluid present in the chamber and / or by introducing gas and / or vapor into the chamber, such as through one or more ports communicating with the formed chamber. For example, a fluid present on a substrate or in a reservoir within the chamber can be heated to increase the pressure within the chamber. In some embodiments, the fluid reservoir is provided within the lower plate or on the substrate itself. In some embodiments, the upper plate itself or a heating element protruding from the upper plate (regardless of its location) can contact the fluid reservoir and heat the fluid in the reservoir to generate vapor. The generated vapor can then be used to heat and / or pressurize the chamber.

[0463] Alternatively, gas and / or vapor can be introduced into the chamber (such as through one or more ports in communication with the chamber's internal environment) to facilitate pressurization and / or heating of the chamber. In some embodiments, the pressure in the chamber is adjusted by turning one or more heating and / or cooling elements off and on, introducing additional gas and / or vapor into the chamber through one or more ports, venting gas and / or vapor through one or more ports, or any combination thereof. In some embodiments, the specimen processing device and / or chamber include one or more safety mechanisms to prevent overpressurization, e.g., a vent port, a force-generating member that can exert a force less than the force exerted outward from internal pressurization of the chamber.

[0464] In some embodiments, the chamber is adapted to maintain a temperature in the range of about 70°C to about 220°C. In other embodiments, the chamber is adapted to maintain a temperature in the range of about 90°C to about 200°C. In still other embodiments, the chamber is adapted to maintain a temperature in the range of about 95°C to about 180°C. In further embodiments, the chamber is adapted to maintain a temperature in the range of about 100°C to about 170°C. In still further embodiments, the chamber is adapted to maintain a temperature in the range of about 110°C to about 160°C. In still further embodiments, the chamber is adapted to maintain a temperature in the range of about 120°C to about 150°C.

[0465] In some embodiments, a pressure in the range of about 95 kPa to about 2000 kPa is maintained in the chamber during heating. In other embodiments, a pressure in the range of about 100 kPa to about 1600 kPa is maintained in the chamber during heating. In still other embodiments, a pressure in the range of about 150 kPa to about 1050 kPa is maintained in the chamber during heating. In further embodiments, a pressure in the range of about 190 kPa to about 850 kPa is maintained in the chamber during heating. In still further embodiments, a pressure in the range of about 260 kPa to about 750 kPa is maintained in the chamber during heating. In still further embodiments, a pressure in the range of about 300 kPa to about 700 kPa is maintained in the chamber during heating.

[0466] In some embodiments, a force-generating member is utilized to exert a force on the chamber, such as a force-generating member that exerts a force on the upper plate via one or more pressure members in communication with the upper plate. In this manner, the force exerted by the force-generating member allows a pressurized internal environment of the chamber to be maintained. Also, as described above, the force exerted by the force-generating member may be predetermined. In some embodiments, the predetermined force exerted by any force-generating member is less than any predetermined threshold pressure. Alternatively, the predetermined force exerted by the force-generating member is programmed not to exceed any predetermined threshold pressure. In this regard, when the pressure in the chamber exceeds a predetermined threshold pressure and the predetermined force is set to be less than or not exceed any predetermined threshold pressure, the force-generating member will be deactivated to allow the chamber to release or vent the excess pressure; for example, the upper and lower plates can at least partially separate from each other to allow gas and / or vapor to be vented to the external environment.

[0467] By way of example, if the chamber or the substrate, fluid, and / or reagents disposed therein are heated to approximately 160° C. and the predetermined threshold pressure is set at 750 kPa, the force-generating member may be configured to apply an amount of force that is less than the predetermined threshold pressure or that does not exceed the predetermined threshold pressure. Continuing this example, if the pressure exceeds the predetermined threshold pressure of 750 kPa (e.g., the chamber reaches a pressure of 760 kPa), the force-generating member may allow the release of excess gas and / or vapor. As described herein, the upper plate and / or lower plate may further include one or more ports and / or valves that allow the release of gas and / or vapor from the chamber when the predetermined threshold pressure is exceeded.

[0468] In some embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 1250 kPa. In other embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 1050 kPa. In still other embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 950 kPa. In further embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 900 kPa. In further embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 850 kPa. In further embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 800 kPa. In yet further embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 750 kPa. In yet further embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 700 kPa. In yet further embodiments, the force-generating member is configured to fail when the pressure in the chamber exceeds 650 kPa. In yet further embodiments, the force-generating member is configured to shut down if the pressure in the chamber exceeds 600 kPa. In still further embodiments, the force-generating member is configured to shut down if the pressure in the chamber exceeds 500 kPa. In yet other embodiments, the force-generating member is configured to shut down if the pressure exceeds 200 kPa, 300 kPa, or 400 kPa. The predetermined threshold pressure at which the force-generating member advances can be set to any value that prevents the chamber pressure from exceeding a value considered unsafe. For example, the predetermined threshold pressure can vary depending on the materials from which the chamber is constructed, local safety regulations, and combinations thereof.

[0469] In some embodiments, one of the lower plate and / or the upper plate can further include one or more temperature and / or pressure sensors. In some embodiments, the one or more temperature sensors are platinum resistance thermometers or thermistors. In other embodiments, the one or more temperature sensors are disposed below the substrate. In other embodiments, the one or more temperature sensors are disposed on the substrate, such as at one end or an edge of the substrate. In some embodiments, the one or more temperature sensors are disposed on a sample disposed on the substrate.

[0470] The complementarity of the shapes and / or sizes of features of the upper and lower plates, and how this complementarity facilitates the formation of a chamber therebetween, is further illustrated in FIGS. 10A through 10C. For example, FIG. 10A illustrates upper plate 30 and lower plate 10, respectively, being integrated and in contact with one another. In some embodiments, upper plate 30 and lower plate 10 contact one another at the interface of lower engagement surface 11 and upper engagement surface 31, respectively. As further illustrated in FIG. 10A, recessed cavity 32 in upper plate 30 is sized to accommodate at least substrate 15 and / or partial substrate stage 12, as well as any analytes, fluids, and / or reagents disposed on the upper surface of substrate 15. In some embodiments, recessed cavity 32 further includes a predetermined amount of headspace, e.g., space surrounding substrate 15. In some embodiments, the chamber is approximately 14 cm. 3 Approximately 25cm from 3 In some embodiments, the chamber has a volume in the range of about 15 cm 3 Approximately 24 cm from 3 In some embodiments, the chamber has a volume in the range of about 16 cm 3 Approximately 23 cm from 3 In some embodiments, the chamber has a volume in the range of about 17 cm 3 Approximately 22 cm from 3 In some embodiments, the chamber has a volume in the range of about 18 cm 3 Approximately 21 cm from 3In some embodiments, the chamber has a volume in the range of about 19 cm 3 Approximately 20cm from 3 It has a volume in the range of

[0471] Similarly, Figure 10B shows the upper plate 30 and lower plate 10, respectively, brought together and in contact with one another, with the upper engagement surface 31 contacting an optional seal (seal not shown) disposed within groove 13 of the lower engagement plate 11 and / or lower plate 10. Additionally, Figure 10B shows a recessed cavity 32 within the body 33 of the upper plate 30, again sized to accommodate at least a portion of the substrate 15 and / or substrate stage 12. In some embodiments, the recessed cavity 32 of Figure 10B includes an additional headspace 40 surrounding the substrate 15, as compared to the recessed cavity 32 of Figure 10A.

[0472] 10C further illustrates that the body 16 of the lower plate 10 may be in communication with a heating element 21, such as any of these types of heating elements described herein. The embodiments illustrated in FIGS. 10A-10C may further include one or more pressure members in communication with the upper plate 30 (e.g., to receive an externally applied force and impart that force to the upper and / or lower plates), one or more passive and / or active cooling elements (e.g., a heat sink 39 in communication with the lower plate 10 to passively dissipate heat, cooling channels in thermal communication with one of the upper and / or lower plates to actively remove heat), and / or one or more heating elements (e.g., one or more heating elements embedded within or in communication with the upper and / or lower plates 30 and / or 10 to heat the upper and lower plates or any material disposed on the surface of a substrate when placed within the formed chamber) (see, e.g., FIG. 10D).

[0473] 10E, in some embodiments, a chamber may be formed from complementary upper and lower plates 30 and 10, respectively, where the lower plate 10 includes a lower engagement surface 11 and a substrate stage 12 recessed relative to the lower engagement surface 11, and the upper plate 30 includes an upper engagement surface 31 but does not include a recessed cavity. FIG. 10F shows an alternative embodiment in which the upper plate 30 includes a cavity recessed relative to the upper engagement surface 31, and the lower plate 10 includes a substrate stage 12 recessed relative to the lower engagement surface 11. When the two plates contact each other, a chamber 32 is formed.

[0474] 11A shows a non-limiting embodiment illustrating the positioning of lower plate 10 and upper plate 30 relative to one another such that complementary features on the respective lower and upper plates can align. In some embodiments, lower plate 10 is positioned such that at least a portion of lower engagement surface 11 of lower plate 10 aligns with a portion of upper engagement surface 31 of upper plate 30. FIG. 11B shows lower plate 10 and upper plate 30 in physical communication with one another, e.g., at least a portion of lower engagement surface 11 of lower plate 10 contacts a portion of upper engagement surface 31 of upper plate 30, thereby forming a chamber enclosing at least a portion of substrate 15 and any analyte, fluid, and / or reagent disposed thereon.

[0475] In some embodiments, the chambers formed from the upper plate 30 and the lower plate 10, respectively, are in communication with one or more heating and / or cooling elements so that any substrate or sample disposed with the chamber can be heated and / or cooled, or so that any fluid present in the chamber (e.g., on the substrate or in a separate reservoir) can be heated to increase the pressure within the chamber. In some embodiments, at least one of the upper plate 30 or the lower plate 10 includes a heating element. In some embodiments, at least one of the upper plate 30 or the lower plate 10 includes a cooling element (e.g., a passive cooling element or an active cooling element). In some embodiments, the cooling element is an active cooling element that utilizes a liquid heat transfer medium. In other embodiments, the cooling element is a passive cooling element, such as a heat sink. In some embodiments, the cooling element is a heat sink coupled to the body of the lower plate. In some embodiments, the cooling element includes a liquid heat transfer medium and a pump or other device for circulating the liquid heat transfer medium (see, e.g., FIG. 21C). In some embodiments, the cooling and heating elements are operated together to provide a uniform or homogeneous temperature throughout the substrate and / or sample (e.g., each may be turned off or on in a controlled manner as needed to provide a substrate or sample with a uniform temperature distribution). In some embodiments, it is believed that a uniform temperature distribution in the sample can be achieved when using a heated surface below the substrate or by introducing an additional heating source above the sample (see Figures 21A and 21B). In some embodiments, the chamber includes one or more pressure and / or temperature sensors.

[0476] 30A through 30D illustrate alternative embodiments of chambers formed from upper and lower plates 30 and 10, respectively, further illustrating the relative positioning of one or more heating and / or cooling elements 21 within the upper and lower plates, as well as the relative positioning of the heating and / or cooling elements with respect to the chamber and / or substrate disposed within the chamber. For example, FIG. 30A illustrates that upper plate 30 can include one or more heating and / or cooling elements 21 embedded within the body 33 of the upper plate, while one or more heating and / or cooling elements 21 are also embedded within the body 16 of the lower plate.

[0477] FIG. 30B shows an embodiment in which the upper and lower plates each include fluid channels (411A to 411D), which are located below the recess 32 and below the substrate stage 12. The fluid channels 411A to 411D can be independently operated. For example, the type of fluid, the flow rate, and the temperature of each channel may be independently controlled. Heating cartridges inserted into the bores 401A and 401B of the lower plate 10 can also be independently controlled and, together with the fluid channels, can be used to adjust the temperature of the substrate stage, the substrate, or any specimens disposed on the substrate. In some embodiments, the heating and / or cooling elements are independently operated to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0478] Figure 30C provides yet another alternative embodiment of a chamber formed from upper and lower plates, where the upper and lower plates include multiple fluid flow paths 411A through 411F. Figure 30D illustrates an embodiment in which the lower plate 16 is similar to that shown in Figure 30B, but the upper plate includes a thermoelectric module 402 to provide heating and / or cooling of the body 33 of the upper plate 30. In some embodiments, as shown in Figures 30E and 30F, the fluid channels in the upper and lower plates 30 and 10, respectively, may be connected so that fluid flows through one fluid channel in the upper or lower plate and then through the other fluid channel in the lower or upper plate.

[0479] In some embodiments, any of the heating and / or cooling elements in the upper plate 30 can be operated in conjunction with the heating and / or cooling elements in the lower plate 10 or those embedded in one or more thermal management modules. As a result, by independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate stage, any portion of the substrate supported by the substrate stage, and / or the chamber can be controlled.

[0480] For example, various heating and / or cooling elements present on the upper and lower plates can be independently operated so that at least a portion of the substrate or a sample disposed thereon remains the coldest structure within the chamber, e.g., the substrate or a specimen disposed thereon is maintained at a lower temperature than the upper plate, the lower plate, ports, valves, and / or any other structures within the chamber formed from the upper and lower plates. In some embodiments, the heating and / or cooling elements are independently operated to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0481] Referring to FIG. 40B , different temperature zones, such as temperature zones A, B, C, D, and E, can be established. In some embodiments, each of these different temperature zones is adjacent to and / or in thermal communication with a portion of the body 16 of the lower plate 10 and / or the body 33 of the upper plate 30. The temperature within each of these zones can be established, maintained, and / or adjusted by independently controlling any number of heating and / or cooling elements in thermal communication with any of the illustrated zones, including heating and / or cooling elements disposed within either the lower plate, the upper plate, or any thermal management module. In some embodiments, at least one of a portion of the substrate 15, the surface of the substrate stage 12C, or the substrate stage itself is maintained at a lower temperature than other portions of the body 16 of the lower plate 10 or other portions of the body 33 of the upper plate 30. In some embodiments, at least a portion of the substrate disposed within the chamber formed from the upper and lower plates is maintained as the coldest component within the chamber, such as during an unmasking operation (see FIGS. 43B through 43G ).

[0482] By way of example, and referring again to FIG. 40B , Zone A can be established at a lower temperature than any of Zones B, C, D, and / or E. In some embodiments, Zones A and B are maintained at approximately the same temperature, while Zones C, D, and E are maintained at a relatively lower temperature. One skilled in the art will appreciate that one or more heating and / or cooling elements disposed proximate Zone B allow Zones A and B to be maintained within a first temperature range, while one or more heating and / or cooling elements disposed proximate each of Zones C, D, and E allow Zones C and D to be maintained at a second temperature range. In some embodiments, the first temperature range is lower than the second temperature range. In some embodiments, the first temperature range is about 2% lower than the second temperature range. In other embodiments, the first temperature range is about 3% lower than the second temperature range. In some embodiments, the first temperature range is about 4% lower than the second temperature range. In some embodiments, the first temperature range is about 5% lower than the second temperature range. In some embodiments, the first temperature range is about 7% lower than the second temperature range. In some embodiments, the first temperature range is about 8% lower than the second temperature range. In some embodiments, the first temperature range is about 10% lower than the second temperature range. In some embodiments, the first temperature range is about 12% lower than the second temperature range. In some embodiments, the first temperature range is about 15% lower than the second temperature range. In some embodiments, the first temperature range is about 20% lower than the second temperature range. In some embodiments, the first temperature range is about 25% lower than the second temperature range. In some embodiments, the first temperature range is about 30% lower than the second temperature range.

[0483] 41A, 41B, and 41C each illustrate a substrate 15 in heat communication with one or more heating and / or cooling elements (see, e.g., heating and / or cooling elements 401A, 401B, 411A, and 411B in FIG. 41B). Each of FIGS. 41A, 41B, and 41C illustrates that individual heating and / or cooling elements can be independently operated such that at least a portion of the substrate (or specimen disposed on the substrate) within the chamber formed by the upper and lower plates 30 and 10, respectively, can have a temperature lower than the temperature of other components disposed within the chamber. In this regard, as discussed above, any condensate formed within the chamber can be driven to the coldest portion of the chamber, e.g., the coldest portion of the specimen or substrate, during any temperature reduction operation. In this manner, evaporated fluids and / or reagents can be returned to the substrate and / or specimen disposed on the substrate.

[0484] Specimen processing

[0485] The specimen processing assembly of the present disclosure is configured to facilitate processing of specimens disposed on a substrate surface, hi some embodiments, specimen processing includes unmasking the specimen, e.g., antigen retrieval and / or target retrieval.

[0486] In some embodiments, different specimen processing operations may occur in the same or different regions of the specimen processing assembly. In some embodiments, each of these different specimen processing regions may be a pre-designated region, e.g., a region reserved with the specimen processing assembly for performing a particular processing operation. FIGS. 13A and 13B provide two non-limiting examples of pre-designated specimen processing regions in the context of a movable lower plate coupled to the subassembly 101. As described further below, the lower plate can be moved along the subassembly 101 to different pre-designated processing regions. As another example, as shown in FIGS. 20A and 20B, one or more dispensers can be positioned above one or more substrate trays or carousels. In this example, the region defined by the one or more substrate trays, carousel, and dispenser is a pre-established specimen preparation region.

[0487] Alternatively, the sample processing region may be defined by the presence or absence of one or more sample processing assembly components. For example, in the case of a sample processing assembly including a fixed lower plate, a user can place a sample-holding substrate on top of the substrate stage of the fixed lower plate, which defines the loading region. One or more dispensers, mixers, and / or liquid removal devices can then be moved to the fixed lower plate to dispense one or more fluids and / or reagents onto the substrate. The presence of one or more dispensers in this region defines the region as a sample preparation region.

[0488] The upper plate (and / or force-generating member) can then be moved to the fixed lower plate (after one or more dispensing devices, mixing devices, and / or liquid removal devices have moved out of the region) to form a chamber between the movable upper plate and the fixed lower plate. The formed chamber then defines the unmasking region. As in this example, any one region can serve multiple purposes; for example, the same region can be used as a preparation region and an unmasking region, depending on which devices and / or components are located in that region.

[0489] As another example, the lower plate may be moved from a first position to a second position. In some embodiments, the first position is within the loading zone, thus constituting a predesignated zone. In some embodiments, the second position may be defined as a preparation zone or an unmasking zone, depending on which components are present with the lower plate. For example, one or more dispensing devices may be moved to the lower plate at the second position, thereby defining the preparation zone. After dispensing one or more fluids and / or reagents onto the sample-holding substrate, the upper plate may be moved to the lower plate at the second position to form chambers. In some embodiments, the formed chambers define the unmasking zone.

[0490] In the context of the sample processing assembly 100 including the lower plate 10 movably coupled to the subassembly 101, the lower plate 10 can be moved from a first position to a second position, a third position, and an nth position. For example, in some embodiments, the lower plate 10 can be moved between a loading area, a preparation area, and a demasking area. Of course, the lower plate 10 may be moved or held in any intermediate area between any of the loading area, preparation area, or demasking area. In some embodiments, the sample-holding substrate is loaded onto the lower plate in the loading area. Alternatively, the sample-holding substrate is loaded onto an intermediate member for holding and / or transporting the substrate within the loading area.

[0491] Examples of intermediate members, such as a substrate loader 302, a substrate holder 310, and / or a substrate tray, are shown in Figures 19A-19C and 20A-20B. In these embodiments, the intermediate members can transfer substrates to the lower plate for further processing. Following loading of the specimen-holding substrates, the specimens can be treated in the preparation area with one or more fluids and / or reagents. An unmasking operation can then be performed at elevated temperatures and / or pressures in the unmasking area (see Figures 43A-43F). For example, once the specimen-holding substrate 15 is moved into position, such as within the chamber formed by the upper plate 30 and the lower plate 10, the unmasking operation can render surface antigens and / or nucleic acids visually distinguishable, regardless of whether a subsequent staining procedure is applied. Unmasking can be performed according to any of the methods described in PCT Publication WO 2013 / 079606, the disclosure of which is incorporated herein by reference in its entirety.

[0492] 13A and 13B show the various specimen processing areas in the context of a movable lower plate. In these embodiments, the lower plate 10 can be moved between the loading area 110, the preparation area 111, and the unmasking area 112. In some embodiments, the lower plate 10 may be moved or held in any intermediate area between any of the loading area 110, the processing area 111, or the unmasking area 112.

[0493] In some embodiments, the lower plate 10 is first moved to a loading area, such as shown in Figures 13A and 13B, if it is not already located in such an area. Once in the loading area, a specimen-holding substrate 15 can be placed on the upper surface 12C of the substrate stage 12 of the lower plate 10, such as by an operator of the specimen processing assembly or a system including the specimen processing assembly. In some embodiments, the specimen-holding substrate 15 is a microscope slide.

[0494] The lower plate 10 can then be moved from the loading area 110 to either the preparation area 111 or the unmasking area 112. In one embodiment, as shown in FIG. 13A , the lower plate 10 is moved from the loading area to the preparation area so that one or more fluids and / or reagents can be deposited or dispensed onto the surface of the substrate 15 and / or any samples disposed thereon. In some embodiments, one or more unmasking agents can be dispensed onto the specimen while in the preparation area, and unmasking can be initiated upon dispensing of such reagents. In some embodiments, the total volume of the one or more fluids and / or reagents dispensed onto the substrate or specimens disposed thereon ranges from about 100 μL to about 2000 μL. In some embodiments, the total volume of the one or more fluids and / or reagents dispensed onto the substrate or specimens disposed thereon ranges from about 100 μL to about 1500 μL. In some embodiments, the total volume of the one or more fluids and / or reagents dispensed onto the substrate or specimens disposed thereon ranges from about 100 μL to about 1250 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed thereon ranges from about 100 μL to about 1000 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed thereon ranges from about 100 μL to about 900 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed thereon ranges from about 150 μL to about 800 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed thereon ranges from about 200 μL to about 750 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed thereon ranges from about 250 μL to about 700 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed thereon ranges from about 250 μL to about 650 μL.In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed on the substrate ranges from about 250 μL to about 600 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed on the substrate ranges from about 250 μL to about 550 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed on the substrate ranges from about 300 μL to about 550 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed on the substrate ranges from about 350 μL to about 550 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto the substrate or specimen disposed on the substrate is about 500 μL.

[0495] Referring again to FIG. 13A , once all fluids and / or reagents have been dispensed onto the analyte-holding substrate, the lower plate 10 can be moved to the unmasking region 11...

Claims

1. (i) at least one demasking chamber having a predetermined internal volume, the at least one demasking chamber comprising an upper plate and a lower plate, the lower plate including a lower engagement surface and one or more substrate stages adapted to hold a substrate horizontally within the at least one demasking chamber, the upper plate including an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface, and at least one of the upper plate and the lower plate including at least one of a heating element or a cooling element; (ii) a dyeing module; A system comprising:

2. The predetermined internal volume is about 14 cm ３ Approximately 25 cm from ３ The system of claim 1 , wherein the range is

3. The predetermined internal volume is about 16 cm ３ Approximately 22 cm from ３ 3. The system according to claim 1, wherein the range is:

4. The predetermined internal volume is about 18 cm ３ About 20 cm from ３ 4. The system according to claim 1, wherein the range is:

5. The system of claim 1 , wherein the one or more substrate stages are raised relative to the lower engagement surface.

6. The system of claim 1 , wherein at least one of the lower plate and / or the upper plate further comprises one or more alignment members.

7. The system of claim 1 , wherein at least one of the lower plate and / or the upper plate further comprises one or more temperature and / or pressure sensors.

8. 8. The system of claim 1, wherein at least one of the lower plate and / or the upper plate further comprises one or more temperature sensors in contact with the horizontally held substrate or a fluid disposed thereon.

9. The system of claim 1 , wherein the top plate comprises one or more steam injection ports.

10. The system of claim 1 , wherein the body of the lower plate and the body of the upper plate comprise complementary wedge shapes.

11. The system of claim 1 , wherein at least one of the lower plate and the upper plate comprises at least one seal.

12. The system of claim 11 , wherein the at least one seal is removable.

13. The system of claim 11 , wherein the bottom plate includes a groove, and the at least one removable seal at least partially engages the groove.

14. The system of claim 12 , wherein the at least one removable seal is integrated into a removable seal attachment, the removable seal attachment engaging a portion of a periphery of the lower plate or the upper plate.

15. 15. The system of claim 1, wherein at least one of the upper plate or the lower plate is coupled to at least one of a motor, a piston, a spring, a screw mechanism, a lever, or a cam mechanism.

16. The system of claim 1 , wherein the upper plate and the lower plate are independently movable.

17. 17. The system of any one of claims 1 to 16, further comprising a control system.

18. 20. The system of claim 17, wherein the control system is adapted to operate the at least one heating or cooling element to uniformly heat and / or cool the one or more substrate stages.

19. 19. The system of any one of claims 1 to 18, further comprising at least one substrate transfer device.

20. 20. The system of claim 19, wherein the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and a carrier transport.

21. 21. The system of any one of claims 1 to 20, further comprising one or more substrate loading stations.

22. 22. The system of any one of claims 1 to 21, wherein the bottom plate is modular.

23. 23. The system of claim 22, wherein the lower plate is modular and transportable to the upper plate.

24. 24. The system of any one of claims 1 to 23, wherein the top plate is coupled to a force-generating member.

25. 25. The system of any one of claims 1 to 24, wherein the force-generating member is configured to disable when pressure in the chamber exceeds a predetermined threshold.

26. 1. A system comprising: (i) at least one demasking chamber having a predetermined internal volume, the at least one demasking chamber comprising an upper plate and a lower plate, the lower plate comprising a lower engagement surface and one or more substrate stages adapted to hold a substrate horizontally within the at least one demasking chamber, the upper plate comprising an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface, the upper plate comprising one or more steam injection ports for introducing steam into the at least one demasking chamber; (ii) a dyeing module; (iii) a vapor reservoir; and A system comprising:

27. 27. The system of claim 26, wherein the top plate is coupled to a force-generating member.

28. 28. The system of claim 27, wherein the force generating member is selected from the group consisting of a motor, a spring, a screw, a lever, a piston, a cam, or any combination thereof.

29. The at least one demasking chamber has a diameter of about 14 cm ３ Approximately 25 cm from ３ 29. The system of any one of claims 26 to 28, comprising a predetermined internal volume in the range of

30. 30. The system of any one of claims 26 to 29, wherein at least one of the lower plate and / or the upper plate further comprises one or more alignment members.

31. 31. The system of any one of claims 26 to 30, wherein at least one of the lower plate and / or the upper plate further comprises one or more temperature and / or pressure sensors.

32. 32. The system of any one of claims 26 to 31, wherein at least one of the bottom plate and the top plate further comprises at least one seal.

33. 33. The system of claim 32, wherein the at least one seal is removable.

34. 34. The system of claim 33, wherein the bottom plate comprises a groove, and the at least one removable seal at least partially engages the groove.

35. 1. A system comprising: (i) at least one specimen processing assembly, the specimen processing assembly comprising: (1) a lower plate having a body, the body comprising: (a) a substrate stage having an upper surface adapted to horizontally support a substrate; (b) a lower engagement surface; and (c) a first lower temperature adjustment element in thermal communication with the substrate stage; and (2) an upper plate having an upper engagement surface complementary to the lower engagement surface; (ii) a staining module.

36. 36. The system of claim 35, wherein the first lower temperature adjustment element is positioned below the substrate stage.

37. 37. The system of claim 35 or 36, wherein the lower plate further comprises a second lower temperature adjustment element and a third lower temperature adjustment element, the second lower temperature adjustment element and the third lower temperature adjustment element being respectively positioned adjacent to the first lower temperature adjustment element.

38. 38. The system of claim 37, wherein the first lower temperature adjustment element has a first heat output, the second lower temperature adjustment element has a second heat output, and the third lower temperature adjustment element has a third heat output, and the first heat output is less than either the second heat output or the third heat output.

39. 39. The system of claim 38, wherein a temperature gradient is maintained between the first and second lower temperature adjustment elements and between the first and third temperature adjustment elements.

40. 40. The system of claim 39, wherein the temperature gradient maintained between the first lower temperature adjustment element and the second lower temperature adjustment element is in the range of 2°C to approximately 10°C, and the temperature gradient maintained between the first lower temperature adjustment element and the second lower temperature adjustment element is in the range of 2°C to approximately 10°C.

41. 41. The system of any one of claims 35 to 40, wherein the bottom plate is modular.

42. 42. The system of claim 41, wherein the lower plate is modular and transportable to the upper plate.

43. 43. The system of claim 42, wherein the top plate includes an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface.

44. 44. The system of claim 42 or 43, wherein the top plate is coupled to a force-generating member.

45. 45. The system of claim 44, wherein the force-generating member is configured to disable when pressure within a chamber formed by the upper plate and the lower plate exceeds a predetermined threshold.

46. 46. ​​The system of any one of claims 35 to 45, wherein the first lower temperature adjustment element in thermal communication with the substrate stage is configured such that the substrate stage or a substrate disposed thereon remains the coldest component within a chamber formed from the upper plate and the lower plate.

47. 47. The system of any one of claims 35 to 46, further comprising at least one substrate transfer device.

48. 48. The system of claim 47, wherein the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and a carrier transport.

49. 49. The system of any one of claims 35 to 48, further comprising one or more substrate loading stations.

50. 50. Use of the system of any one of claims 1 to 49 in the preparation of an unmasked specimen.

51. 51. Use of the system of claim 50, wherein the specimen is a histological sample.

52. 51. Use of the system of claim 50, wherein the specimen is a cytological sample.

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